PROCESS FOR THE PRODUCTION OF POLYUNSATURATED FATTY ACIDS IN TRANSGENIC PLANTS

Abstract

Process for the production of polyunsaturated fatty acids in transgenic plants Abstract The present invention relates to a process for the production of polyunsaturated fatty acids in the seed of transgenic plants for introducing, into the organism, nucleic acids which encode polypeptides with eo3-desaturase, A12-desaturase, A6-desaturase, A6-elongase, A5-desaturase, A5-elongase and/or A4-desaturase activity, preferably polypeptides with A6-desaturase, A6-elongase and A5-desaturase activity. The nucleic acid sequences are the sequences shown in SEQ ID NO: 11, SEQ ID NO: 27, SEQ ID NO: 193, SEQ ID NO: 197, SEQ ID NO: 199 and SEQ ID NO: 201. These nucleic acid sequences can advantageously be expressed in the organism, if appropriate together with further nucleic acid sequences which encode polypeptides of the biosynthesis of the fatty acid or iipid metabolism. Especially advantageous are nucleic acid sequences which encode a A6-desaturase, a A5~desaturase, A4-desaturase, A12-desaturase and/or A6-elongase activity. These desaturases and elongases originate advantageously from Thalassiosira, Euglena or Ostreococcus. Furthermore, the invention relates to a process for the production of oils and/or triacylglycerides with an elevated content of long-chain polyunsaturated fatty adds. In a preferred embodiment, the invention furthermore relates to a process for the production of arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid and to a process for the production of triglycerides with an elevated content of unsaturated fatty acids, in particular arachidonic acid, eicosapentaenoic acid and/or docosahexaenoic acid, in transgenic plants, advantageously in the seed of the transgenic plant. The invention relates to the generation of a transgenic plant with an elevated content of polyunsaturated fatty acids, in particular arachidonic acid, eicosapentaenoic acid and/or docosahexaenoic acid, as the result of the expression of the elongases and desaturases used in the process according to the invention. The invention furthermore relates to recombinant nucleic acid molecules comprising the nucleic acid sequences which encode the polypeptides with A6-desaturase, A6-elongase, A5-desaturase and A5-elongase activity, either jointly or individually, and transgenic plants which comprise the abovementioned recombinant nucleic acid molecules. A further part of the invention relates to oils, lipids and/or fatty acids which have been produced by the process according to the invention, and to their use. Moreover, the invention relates to unsaturated fatty acids and to triglycerides with an elevated content of unsaturated fatty acids and to their use.

Full Text

Process for the production of polyunsatu rated fatty acids in transgenic plants
The present invention relates to a process for the production of polyunsaturated fatty acids in the seed of transgenic plants by introducing, into the organism, nucleic acids which encode polypeptides with ω3-desaturase, ∆12-desaturase, ∆6-desaturase, ∆6-elongase, ∆5-desaturase, ∆5-elongase and/or ∆4-desaturase activity, preferably polypeptides with ∆6-desaturase, ∆6-elongase and ∆5-desaturase activity.
The nucleic acid sequences are the sequences shown in SEQ ID NO: 11, SEQ ID NO: 27, SEQ ID NO: 193, SEQ ID NO: 197, SEQ ID NO: 199 and SEQ ID NO: 201. Preferably, a further nucleic acid sequence which encodes a polypeptide with a ∆12-desaturase activity is additionally introduced into the plant, in addition to these nucleic acid sequences, and also expressed simultaneously. Especially preferably, this is the nucleic acid sequence shown in SEQ ID NO: 195.
These nucleic acid sequences can advantageously be expressed in the organism, if appropriate together with further nucleic acid sequences which encode polypeptides of the biosynthesis of the fatty acid or liµl d metabolism. Especially advantageous are nucleic acid sequences which encode a ∆6-desaturase, a ∆5-desaturase, ∆4-desaturase, ∆12-desaturase and/or ∆6-elongase activity. These desaturases and elongases originate advantageously from Thalassiosira, Euglena or Ostreococcus. Furthermore, the invention relates to a process for the production of oils and/or triacylglycerides with an elevated content of long-chain polyunsaturated fatty acids.
In a preferred embodiment, the invention furthermore relates to a process for the production of arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid and to a process for the production of triglycericles with an elevated content of unsaturated fatty acids, in particular arichidonic acid, eicosapentaenoic acid and/or docosahexaenoic acid, in transgenic plants, advantageously in the seed of the transgenic plant. The invention relates to the generation of a transgenic plant with an elevated content of polyunsaturated fatty acids, in particular arichidonic acid, eicosapentaenoic acid and/or docosahexaenoic acid, as the result of the expression of the elongases and desaturases used in the process according to the invention.
The invention furthermore relates to recombinant nucleic acid molecules comprising the nucleic acid sequences which encode the polypeptides with ∆6-desaturase, ∆6-elongase, ∆5-desaturase and ∆5-elongase activity, either jointly or individually, and

transgenic plants which comprise the abovementioned recombinant nucleic acid molecules.
A further part of the invention relates to oils, liµl ds and/or fatty acids which have been produced by the process according to the invention, and to their use. Moreover, the invention relates to unsaturated fatty acids and to triglycerides with an elevated content of unsaturated fatty acids and to their use.
Liµl d synthesis can be divided into two sections: the synthesis of fatty acids and their binding to sn-giycerol-3-phosphate, and the addition or modification of a polar head group. Usual liµl ds which are used in membranes comprise phospholiµl ds, glycoliµl ds, sphingoliµl ds and phosphoglycerides. Fatty acid synthesis starts with the conversion of acetyl-CoA into malonyl-CoA by acetyl-CoA carboxylase or into acetyl-ACP by acetyl transacylase. After condensation reaction, these two product molecules together form acetoacetyl-ACP, which is converted via a series of condensation, reduction and dehydration reactions so that a saturated fatty acid molecule with the desired chain length is obtained. The production of the unsaturated fatty acids from these molecules is catalyzed by specific desaturases, either aerobically by means of molecular oxygen or anaerobically (regarding the fatty acid synthesis in microorganisms, see F.C. Neidhardt et al. (1996) E. coli and Sal.,onella. ASM Press: Washington, D.C., p. 612-636 and references cited therein; Lengeler et al. (Ed.) (1999) Biology of Procaryotes. Thieme: Stuttgart, New York, and the references therein, and Magnuson, K.f et al. (1993) Microbiological Reviews 57:522-542 and the references therein). To undergo the further elongation steps, the resulting phospholiµl d-bound fatty acids must be returned to the fatty acid CoA ester pool. This is made possibly by acyl-CoA:lysophospholiµl d acyltransferases. Moreover, these enzymes are capable of transferring the elongated fatty acids from the CoA esters back to the phospholiµl ds. If appropriate, this reaction sequence can be followed repeatedly.
Furthermore, fatty acids must subsequently be transported to various modification sites and incorporated into the triacylglycerol storage liµl d. A further important step during liµl d synthesis is the transfer of fatty acids to the polar head groups, for example by glycerol fatty acid acyltransferase (see Frentzen, 1998, Liµl d, 100(4-5): 161-166).
With regard to publications on the biosynthesis of fatty acids in plants, desaturation, the liµl d metabolism and the membrane transport of liµl dic compounds, beta-oxidation, the modification of fatty acids and cofactors and the storage and assembly of triacylglycerol, including the references cited therein, see the following papers: Kinney,

derived from arachidonic acid and eicosapentaenoic acid. Eicosanoids (known as the PG2 series) which are formed from the ω6-atty acids, generally promote inflammatory reactions, while eicosanoids (known as the PG3 series) from ω3--fatty acids have little or no proinflammatory effect.
Polyunsaturated long-chain ω3-fatty acids such as eicosapentaenoic acid (= EPA, C20:5∆5.8,11,14,17) or docosahexaenoic acid (= DHA, C22:6∆4,7,10-13,16,19) are important, components of human nutrition owing to their various roles in health aspects, including the development of the child brain, the functionality of the eyes, the synthesis of hormones and other signal substances, and the prevention of cardiovascular disorders, cancer and diabetes (Poulos, A Liµl ds 30:1-14, 1995; Horrocks, LA and Yeo YK Pharmacol Res 40:211-225, 1999). There is therefore a demand for the production of polyunsaturated long-chain fatty acids.
Owing to the present-day composition of human food, an addition of polyunsaturated ω3--fatty acids, which are preferentially found in fish oils, to the food is particularly important. Thus, for example, polyunsaturated fatty acids such as docosahexaenoic acid (= DHA, C22:6∆5,8,14,11,17) or eicosapentaenoic acid (= EPA, C20:5∆5'8'11iW7) are added to infant formula to improve the nutritional value. The unsaturated fatty acid DHA is supposed to have a positive effect on the deveopment and maintenance of brain function. There is therefore a demand for the production of polyunsaturated long-chain fatty acids.
The various fatty acids and triglycerides are mainly obtained from microorganisms such as Mortierella or Schizochytrium or from oil-producing plants such as soybeans, oilseed rape, algae such as Crypthecodinium or Phaeodactylurn and others, being obtained, as a rule, in the form of their triacylglycerides (= triglycerides = triglycerols). However, they can also be obtained from animals, for example, fish. The free fatty acids are advantageously prepared by hydrolysis. Very long-chain polyunsaturated fatty acids such as DHA, EPA, arachidonic acid (ARA, C20:4∆5'61114) dihomo-y-linolenic acid (C20:3∆8,11',4) or docosapentaenoic acid (DPA, C22:5∆710'13,16,19) are, however, not synthesized in oil crops such as oilseed rape, soybeans, sunflowers and safflower Conventional natural sources of these fatty acids are fish such as herring, sal.,on, sardine, redfish, eel, carp, trout, halibut, mackerel, zander or tuna, or algae.
Depending on the intended use, oils with saturated or unsaturated fatty acids are preferred, in human nutrition, for example, liµl ds with unsaturated fatty acids, specifically polyunsaturated fatty acids, are preferred. The polyunsaturated tu3~fatty acids are said to have a positive effect on the cholesterol level in the blood and thus on
0

the possibility of preventing heart disease. The risk of heart disease, stroke or hypertension can be reduced markedly by adding these ω3-fatty acids to the food. Also, ω3--fatty acids have a positive effect on inflammatory, specifically on chronically inflammatory, processes in association with immunoiogical diseases such as rheumatoid arthritis. They are therefore added to foodstuffs, specifically to dietetic foodstuffs, or are employed in medicaments. ω3-fatty acids such as arachidonic acid tend to have an adverse effect on these disorders in connection with these rheumatic diseases on account of our usual dietary intake.
Owing to their positive characteristics, there has been no lack of attempts in the past to make available genes which are involved in the synthesis of these fatty acids or triglycerides for the production of oils in various organisms with a modified content of unsaturated fatty acids. Thus, WO 91/13972 and its US equivalent describe a ∆9-desaturase. WO 93/11245 claims a A15-desaturase and WO 94/11516 a ∆12-desaturase. Further desaturates are described, for example, in EP-A-0 550 162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al., J. Biol. Chem., 265, 1990: 20144-20149, Wada et al., Nature 347,1990: 200-203 or Huang et al., Liµl ds 34, 1999: 649-659. However, the biochemical characterization of the various desaturases has been insufficient to date since the enzymes, being membrane-bound proteins, present great difficulty in their isolation and characterization (McKeon et al., Methods in Enzymol. 71, 1981: 12141-12147, Wang et al., Plant Physiol. Biochem., 26, 1988: 777-792). As a rule, membrane-bound desaturases are characterized by being introduced into a suitable organism which is subsequently analyzed for enzyme activity by analyzing the starting materials and the products. ∆6-Desaturases are described in WO 93/06712, US 5,614,393, US5614393 WO 96/21022, WO 00/21557 and WO 99/27111. The application of this enzyme for the production of fatty acids in transgenic organisms is described in WO 98/46763, WO 98/46764 and WO 98/46765. The expression of various desaturases is described and claimed in WO 99/64616 or WO 98/46776. As regards the expression efficacy of desaturases and its effect on the formation of polyunsaturated fatty acids, it must be noted that the expression of a single desaturase as described to date has only resulted in low contents of unsaturated fatty acids/liµl ds such as, for example, ylinolenic acid and stearidonic acid.
There have been a number of attempts in the past to obtain elongase genes. Millar and Kunst, 1997 (Plant Journal 12:121-131) and Millar et al., 1999 (Plant Cell 11:825-838) describe the characterization of plant elongases for the synthesis of monounsaturated long-chain fatty acids (C22:1) and for the synthesis of very long-chain fatty acids for the

formation of waxes in plants (C28-C32). The synthesis of arachidonic acid and EPA is described, for example, in WO 01/59128, WO 00/12720, WO 02/077213 and WO 02/08401. The synthesis of polyunsaturated C24-fatty acids is described, for example, in Tvrdik et al. 2000, J. Cell Biol. 149:707-718 or WO 02/44320.
Especially suitable microorganisms for the production of PUFAs are microorganisms such as microalgae such as Phaeodactylum tricornutum, Porphiridium species, Thraustochytrium species, Schizochytrium species or Crypthecodinium species, ciliates such as Stylonychia or Colµl dium, fungi such as Mortierella, Entomophthora or Mucor and/or mosses such as Physcomitrella, Ceratodon and Marchantia (R. Vazhapµl lly & F Chen (1998) Botanica Marina 41: 553-558; K. Totani & K. Oba (1987) Liµl ds 22: 1060-1062; M. Akimoto et al. (1998) Appl. Biochemistry and Biotechnology 73: 269-278). Strain selection has resulted in the development of a number of mutant strains of the microorganisms in question which produce a series of desirable compounds including PUFAs. However, the mutation and selection of strains with an improved production of a particular molecule such as the polyunsaturated fatty acids is a time-consuming and difficult process, which is why as described above recombinant methods are preferred However, only limited amounts of the desired polyunsaturated fatty acids such as DPA, EPA or ARA can be produced with the aid of the abovementioned microorganisms; where, as a rule, they are generally obtained as fatty acid mixtures, depending on the microorganisms used.
Higher plants comprise polyunsaturated fatty acids such as linoleic acid (C18:2) and linolenic acid (C18:3). ARA, EPA and DHA are found not at all in the seed oil of higher plants, or only in miniscule amounts (E. Ucciani: Nouveau Dictionnaire des Huiles Vegetales [New Dictionary of the Vegetable Oils]. Technique & Documentation -Lavoisier, 1995. ISBN: 2-7430-0009-0). However, the production of LCPUFAs in higher plants, preferably in oilseed crops such as oilseed rape, linseed, sunflowers and soybeans, would be advantageous since large amounts of high-quality LCPUFAs for the food industry, animal nutrition and pharmaceutical purposes might be obtained economically. To this end, it is advantageous to introduce, into oilseed crops, genes which encode enzymes of the LCPUFA biosynthesis via recombinant methods and to express them therein. These genes encode for example ∆6-desaturases, ∆6-elongases, ∆5-desaturases, ∆5-elongases or ∆4-desaturases. These genes can advantageously be isolated from microorganisms and lower plants which produce LCPUFAs and incorporate them in the membranes or triacylglycerides. Thus, it has already been possible to isolate ∆6-desaturase genes from the moss Physcomitrella patens and ∆6-elongase genes from P. patens and from the nematode C. elegans.

A variety of synthetic pathways is being discussed for the synthesis of arachidonic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Figure 1). Thus, EPA or DHA are produced in marine bacteria such as Vibrio sp. or Shewanella sp. via the polyketide pathway (Yu, R. et al. Liµl ds 35:1061-1064, 2000; Takeyama, H. et al. Microbiology 143:2725-2731, 1997).
An alternative strategy is the alternating activity of desaturases and elongases (Zank, T.K. et al. Plant Journal 31:255-268, 2002; Sakuradani, E. et al. Gene 238:445-453, 1999). A modification of the above-described pathway by ∆6-desaturase, ∆6-elongase, ∆5-desaturase, ∆5-elongase and ∆4-desaturase is the Sprecher pathway (Sprecher 2000, Biochim. Biophys. Acta 1486:219-231) in mammals. Instead of the ∆4-desaturation, a further elongation step is effected here to give C24, followed by a further ∆6-desaturation and finally p-oxidation to give the C22 chain length. Thus what is known as Sprecher pathway (see Figure 1) is, however, not suitable for the production in plants and microorganisms since the regulatory mechanisms are not known.
Depending on their desaturation pattern, the polyunsaturated fatty acids can be divided into two large classes, viz. co6- or ω3-fatty acids, which differ with regard to their metabolic and functional activities (Fig. 1).
The starting material for the ω6--metabolic pathway is the fatty acid linoleic acid (18:2A9*12) while the ω3--pathway proceeds via linolenic acid (18:3∆9'12'15). Linolenic acid is formed by the activity of an ω3-desaturase (Tocher et al. 1998, Prog. Liµl d Res. 37 73-117; Domergueetal. 2002, Eur. J. Biochem. 269, 4105-4113).
Mammals, and thus also humans, have no corresponding desaturase activity (∆12- and ω3-desaturase) and must take up these fatty acids (essential fatty acids) via the food Starting with these precursors, the physiologically important polyunsaturated fatty acids arachidonic acid (= ARA, 20:4∆5i8f11t14), an w6-fatty acid and the two ω3-fatty acids eicosapentaenoic acid (= EPA, 20:5∆5,8,114,17) and docosahexaenoic acid (DHA, 22:6∆4l7i10'13'17r19) are synthesized via the sequence of desaturase and elongase reactions. The application of ω3-fatty acids shows the therapeutic activity described above in the treatment of cardiovascular diseases (Shimikawa 2001, World Rev. Nutr. Diet. 88, 100-108), inflammations (Calder 2002, Proc. Nutr. Soc. 61, 345-358) and arthritis (Cleland and James 2000, J. Rheumatol. 27, 2305-2307).
From the angle of nutritional physiology, it is therefore advantageous to achieve a shift

between the ω6--synthetic pathway and the ω3-synthetic pathway (see Figure 1) so that more ω3-fatty acids are produced. The enzymatic activities of various ω3-desaturases which desaturate C18;2-, C22:4- or C22:5-fatty acids have been described in the literature (see Figure 1). However, none of the desaturases whose biochemistry has been described converts a broad range of substrates of the w6-synthetic pathway into the corresponding fatty acids of the ω3--synthetic pathway.
The elongation of fatty acids, by elongases, by 2 or 4 C atoms is of crucial importance for the production of C20- and C22-PUFAs, respectively. This process proceeds via 4 steps. The first step is the condensation of malonyl-CoA onto the fatty-acid-acyl-CoA by ketoacyl-CoA synthase (KCS, hereinbelow referred to as elongase). This is followed by a reduction step (ketoacyl-CoA reductase, KCR), a dehydratation step (dehydratase) and a final reduction step (enoyl-CoA reductase). It has been postulated that the elongase activity affects the specificity and rate of the entire process (Millar and Kunst, 1997 Plant Journal 12:121-131).
No specific elongase has been described to date for the production of DHA (C22:6 n-3) in organisms which do not naturally produce this fatty acid. Only elongases which provide C20- or C24-fatty acids have been described to date. A ∆5-elongase activity has not been described to date.
The first transgenic plants which comprise and express genes encoding LCPUFA biosynthesis enzymes and which, as a consequence, produce LCPUFAs were described for the first time, for example, in DE-A-102 19 203 (Process for the production of polyunsaturated fatty acids in plants) or in WO 2004/071467. However, these plants produce LCPUFAs in amounts which require further optimization for processing the oils which are present in the plants. Thus, ARA content in the plants described in DE-A-102 19 203 only amounts to 0.4 to 2% and the EPA content only to 0.5 to 1%, in each case based on the total liµl d content of the plants. WO 2004/071467 discloses higher contents of polyunsaturated C20- and C22-fatty acids such as ARA, EPA or DHA. However, the process disclosed has a series of grave disadvantages. It seems that DHA cannot be detected at all in the seeds in the process disclosed. To produce PUFAs, soybean is less suitable, owing to its low oil content of approximately only 20% by weight. Soybean is an advantageous protein source and is therefore grown on a large scale. However, the oil content of soybeans is rather low. Moreover, the dihomo-y-linolenic acid (=DGHL or HGLA) content obtained in the production process is much too high. HGLA is hardly detectable in fish oils or algal oils or microbial oils. A further disadvantage is that the plants disclosed in WO 2004/071467

were generated by cotransformation, which leads to the segregation of the characteristics in the subsequent generations, and thus to an increased selection effort.
To make possible the fortification of food and/or of feed with these polyunsaturated fatty acids, there is therefore a great need for a simple, inexpensive process for the production of these polyunsaturated fatty acids in plant systems, especially in the seed of transgenic plants.
The object of the invention was therefore to develop a process for the production of large amounts of polyunsaturated fatty acids, specifically ARA, EPA and DHA, in the seed of a transgenic plant. This object was achieved by the process according to the invention for the production of compounds of the general formula I

in the seeds of transgenic plants with a content of at least 20% by weight based on the total liµl d content, which comprises the following process steps:
a) introducing, into the organism, at least one nucleic acid sequence which encodes a ∆9-elongase and ∆6-desaturase activity, and
b) introducing, into the organism, at least one nucleic acid sequence which encodes a ∆8-desaturase and ∆6-elongase activity, and
c) introducing, into the organism, at least one nucleic acid sequence which encodes a ∆5-desaturase activity, and
d) introducing, into the organism, at least one nucleic acid sequence which encodes a ∆5-elongase activity, and
e) introducing, into the organism, at least one nucleic acid sequence which encodes a ∆4-desaturase activity, and
where the variables and substituents in formula I have the following meanings: R1 = hydroxyl, coenzyme A (thioester), lysophosphatidylcholine,

n-nonylcarbonyl, n-decylcarbonyl, n-unclecylcarbonyl, n-dodecylcarbonyl, n-tridecyl-carbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyi, n-hepta-decylcarbonyl, n-octadecylcarbonyl-, n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl- or n-tetracosanylcarbonyl, which comprise one or more double bonds. Saturated or unsaturated C10-C22-alkyIcarbonyl radicals such as n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-heptadecylcarbonyl, n-octadecylcarbonyl, n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl or n-tetracosanylcarbonyl, which comprise one or more double bonds are preferred. Especially preferred are saturated and/or unsaturated C10-C22-alkylcarbonyl radicals such as C10-alkylcarbonyl, Cn-alkylcarbonyl, C12-alkylcarbonyl, C13-alkylcarbonyl, C14-alkylcarbonyl, C16-alkylcarbonyl, C18-alkylcarbonyl, C20-alkylcarbonyl or C22-alkylcarbonyl radicals which comprise one or more double bonds. Very especially preferred are saturated or unsaturated Ci6-C22-alkylcarbonyl radicals such as C16-alkylcarbonyl, C18-alkylcarbonyl, C20-alkylcarbonyl or C22-alkylcarbonyl radicals which comprise one or more double bonds. These advantageous radicals can comprise two, three, four, five or six double bonds. The especially preferred radicals with 20 or 22 carbon atoms in the fatty acid chain comprise up to six double bonds, advantageously three, four, five or six double bonds, especially preferably four, five or six double bonds, very especially preferably five or six. All the abovementioned radicals are derived from the corresponding fatty acids.
The abovementioned radicals of R1, R2 and R3 can be substituted by hydroxyl and/or epoxy groups and/or can comprise triple bonds.
The polyunsaturated fatty acids produced in the process according to the invention advantageously comprise at least two, advantageously three, four, five or six, double bonds. The fatty acids especially advantageously comprise four, five or six double bonds. Fatty acids produced in the process advantageously have 18, 20 or 22 C atoms in the fatty acid chain; the fatty acids preferably comprise 20 or 22 carbon atoms in the fatty acid chain. Saturated fatty acids are advantageously reacted to a minor degree, or not at all, by the nucleic acids used in the process. To a minor degree is to be understood as meaning that the saturated fatty acids are reacted with less than 5% of the activity, advantageously less than 3%, especially advantageously with less than 2%, very especially preferably with less than 1, 0.5, 0.25 or 0.125% of the activity in comparison with polyunsaturated fatty adds. These fatty acids which have been produced can be produced in the process as a single product or be present in a fatty acid mixture.

independently of one another are saturated or unsaturated C18-C22-alkylcarbonyl; especially advantageously, are independently of one another C18-, C20- or C22-alkylcarbonyl with at least two double bonds, advantageously with at least three, four, five or six double bonds, especially advantageously with at least four, five or six double bonds.
In a preferred embodiment of the process, a nucleic acid sequence which encodes polypeptides with ω3-desaturase activity, selected from the group consisting of:
a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 87 or SEQ ID NO: 105, or
b) nucleic acid sequences which can be derived form the amino acid sequence shown in SEQ ID NO: 88 or SEQ ID NO: 106 as the result of the degeneracy of the genetic code, or
c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 87 or SEQ ID NO: 105, which encode polypeptides with at least 60% identity at the amino acid level with SEQ ID NO: 88 or SEQ ID NO: 106 and which have ω3-- desaturase activity
is additionally introduced into the transgenic plant.
In a further preferred embodiment of the process, that a nucleic acid sequence which encodes polypeptides with ∆12-desaturase activity, selected from the group consisting of:
a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 107, SEQ ID NO: 109 or SEQ ID NO: 195, or
b) nucleic acid sequences which, as the result of the degeneracy of the genetic code, can be derived from the amino acid sequence shown in SEQ ID NO: 108, SEQ ID NO: 110 or SEQ ID NO: 196, or
c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 107, SEQ ID NO: 109 or SEQ ID NO: 195, which encode polypeptides with at least 60% at the amino acid level with SEQ ID NO: 108, SEQ ID NO: 110 or SEQ ID NO: 196 and which have ∆12-desaturase activity

is additionally introduced into the transgenic plant.
These abovementioned ∆12-desaturase sequences can be used alone or in combination with ω3-desaturase sequences together with the nucleic acid sequences used in the process which encode ∆9-elongases, ∆6-desaturases, ∆8-desaturases, ∆6-elongases, ∆5-desaturases, ∆5-elongases or ∆4-desaturases.
Table 1 shows the nucleic acid sequences, the organism of origin and the sequence ID
number
*

In a further embodiment of the invention, a process to be developed for the production of large amounts of polyunsaturated fatty acids, specifically ARA and EPA, in a transgenic plant. This process is also suitable for the production of DHA. Thus, ARA, EPA, DHA or their mixtures can be produced in the process. A further embodiment of the invention is thus a process for the compounds of the general formula I

in transgenic plants, the process comprising:
a) introducing, into a plant, at least one nucleic acid sequence which encodes a polypeptide with a ∆6-desaturase activity and is selected from the group consisting of:

i) a nucleic acid with the sequence shown in SEQ ID NO: 193 or SEQ ID NO:
201, ii) nucleic acid sequences which encode the amino acid sequence shown in
SEQ ID NO: 194 or SEQ ID NO: 202, iii) nucleic acid sequences which hybridize under stringent conditions with the
complementary strand of the nucleic acid sequence shown in SEQ ID NO:
193 or SEQ ID NO: 201, and iv) nucleic acid sequences which have at least 60% identity with the sequence
shown in SEQ ID NO: 193 or SEQ ID NO: 201,
b) introducing, into a plant, at least one nucleic acid sequence which encodes a polypeptide with a ∆6-elongase activity and is selected from the group consisting of:
i) a nucleic acid with the sequence shown in SEQ ID NO: 27 or SEQ ID NO:
199, ii) nucleic acid sequences which encode the amino acid sequence shown in
SEQ ID NO: 28 or SEQ ID NO: 200, iii) nucleic acid sequences which hybridize under stringent conditions with the
complementary strand of the nucleic acid sequence shown in SEQ ID NO:
27 or SEQ ID NO: 199, and iv) nucleic acid sequences which have at least 60% identity with the sequence
shown in SEQ ID NO: 27 or SEQ ID NO: 199,
c) introducing, into a plant, at least one nucleic acid sequence which encodes a polypeptide with a ∆5-desaturase activity and is selected from the group consisting of:
i) a nucleic acid with the sequence shown in SEQ ID NO: 11,
ii) nucleic acid sequences which encode the amino acid sequence shown in
SEQ ID NO: 12, iii) nucleic acid sequences which hybridize under stringent conditions with the
complementary strand of the nucleic acid sequence shown in SEQ ID NO:
11, and iv) nucleic acid sequences which have at least 60% identity with the sequence
shown in SEQ ID NO: 11, where the variables and substituents in the formula I have the meaning given above.

The nucleic acid sequences which can be used in the process are described in WO 02/26946 (∆5-desaturase from Thraustochytrium ssp., SEQ ID NO: 11 and ∆6-desaturase from Phytium irregulare, SEQ ID NO: 193) and in WO 01/59128 (∆6-elongase from Physcomitrella patens, SEQ ID NO: 27), which is expressly referred to here. However, in these cases, the formation of ARA and EPA was studied either not in transgenic plants, but only in microorganisms, or else no increase ARA and EPA synthesis was detected in the transgenic plants. Moreover, the nucleic acids according to the invention were not combined, in these applications, with nucleic acids which encode other enzymes of the fatty acid biosynthetic pathway.
Surprisingly, it has now been found that the coexpression of the nucleic acids with the sequences shown in SEQ ID NO: 11, 27, 193,199 and 201 leads, in transgenic plants, to a greatly increased ARA content to up to more than 8%, advantageously up to more than 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, especially advantageously to more than 21%, 22%, 23%, 24% or 25%, based on the total liµl d content of the plant (cf. Table 2, Table 3, Table 4 and Figure 31). The abovementioned percentages are percent by weight.
To further increase the yields in the process described for the production of oils and/or triglycerides with a content of polyunsaturated fatty acids, especially ARA, EPA or DHA or their mixtures, which is advantageously increased in comparison with oils and/or triglycerides from wild-type plants, it may be advantageous to increase the amount of the starting material for the fatty acid biosynthesis. This can be achieved for example by introducing a nucleic add which encodes a polypeptide with the activity of a ∆12-desaturase, and coexpressing it in the organism.
This is especially advantageously in oil-producing organisms such as the family Brassicaceae, such as the genus Brassica, for example oilseed rape, turnip rape or Indian mustard; the family Elaeagnaceae, such as the genus Elaeagnus, for the example the genus and species Olea europaea or the family Fabaceae, such as the genus Glycine, for example the genus and species Glycine max, which has a high oleic acid content, but only a low linoleic acid content (Mikoklajczak et al., Journal of the American Oil Chemical Society, 38, 1961, 678-681).
This is why, in a preferred embodiment of the present invention, a nucleic acid sequence which encodes a polypeptide with ∆12-desaturase activity is additionally introduced into the transgenic plant.

Especially preferably, this nucleic acid sequence is selected from the group consisting of:
a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 195,
b) nucleic acid sequences which encode the amino acid sequence shown in SEQ ID NO: 196,
c) nucleic acid sequences which hybridize under stringent conditions with the complementary strand of the nucleic acid sequence shown in SEQ ID NO: 195, and
d) nucleic acid sequences which have at least 60% identity with the sequence shown in SEQ ID NO: 195.
The nucleic acid sequence with the SEQ ID NO: 195 is derived from Calendula officinalis and described in WO 01/85968, the disclosure of which is likewise incorporated in the present application by reference.
The ∆12-desaturases used in the process according to the invention advantageously convert oleic acid (C18:1∆9) into linoleic acid (C18:2∆9'12) or C18:2∆6'9 into C18:3∆8,9,12 (gamma-linolenic acid = GLA), the starting materials for the synthesis of ARA, EPA and DHA. The ∆12-desaturases advantageously convert fatty acids bound to phospholiµl ds or CoA-fatty acid esters, advantageously bound to CoA-fatty acid esters. If an elongation step has taken place beforehand, this advantageously leads to higher yields of synthetic products since, as a rule, elongation takes place at CoA-fatty acid esters, while desaturation predominantly takes place at the phospholiµl d or at the triglycerides. An exchange between the CoA-fatty acid esters and the phospholiµl ds or triglycerides, which would require a further, potentially limiting, enzyme reaction, is thus not required.
The additional expression of the ∆12-desaturase in the transgenic plants leads to a further increase in the ARA content up to more than 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% and 20%, especially advantageously to more than 21%, 22%, 23%, 24% or 25%, based on the total liµl d content of the plant (cf. Tables 3 and 4 and Figure 32). The abovementioned percentages are percent by weight.
Further nucleic acid sequences which encode a polypeptide with a ∆5-elongase activity can advantageously be introduced into the plants in the process according to the

In a further advantageous embodiment of the process, all nucleic acid sequences are introduced into the plants on a shared recombinant nucleic acid molecule, it being possible for each nucleic acid sequence to be under the control of its own promoter and it being possible for this own promoter to take the form of a seed-specific promoter.
However, it is not only the nucleic acids detailed in the sequence listing which can successfully be employed in the invention to carry out the conversion; rather, even sequences which deviate to a certain degree from these sequences and which encode proteins with the essentially identical enzymatic activity can be employed. These take the form of nucleic acids which have a certain degree of identity or homology with the sequences specified in the sequence listing. An essentially identical enzymatic activity denotes proteins which have at least 20%, 30%, 40%, 50% or 60%, advantageously at least 70%, 80%, 90% or 95%, especially advantageously at least 96%, 97%, 98% or 99% of the enzymatic activity of the wild-type enzymes.
In order to determine the percentage of homology (= identity) of two amino acid sequences or of two nucleic acids, the sequences are written one under the other (for example, gaps may be introduced into the sequence of a protein or of a nucleic acid in order to generate optimal alignment with the other protein or the other nucleic acid). Then, the amino acid radicals or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If a position in a sequence is occuµl ed by the same amino acid radical or the same nucleotide as the corresponding position in the other sequence, then the molecules are homologous at this position (i.e. amino acid or nucleic acid "homology" as used in the present context corresponds to amino acid or nucleic acid "identity"). The percentage of homology between the two sequences is a function of the number of positions which the sequences share (i.e. % homology = number of identical positions/total number of positions x 100). The terms homology and identity are therefore to be considered as synonymous.
The homology was calculated over the entire amino add or nucleic acid sequence region. To compare various sequences, the skilled worker has available a series of programs which are based on various algorithms. The algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. The program µl leUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the programs Gap and BestFit [Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970) and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981)], which are part of the GCG software packet [Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991)], were used to carry out the sequence comparisons. The

sequence homology data given above in percent were determined over the entire sequence region using the program GAP with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000. Unless otherwise specified, these settings were always used as standard settings for sequence comparisons.
The skilled worker will recognize that DNA sequence polymorphisms which lead to modifications of the amino acid sequence of SEQ ID NO: 12, 28, 194, 196,198, 200 and/or 202 may occur within a population. These natural variants usually cause a variance of from 1 to 5% in the nucleotide sequence of the ∆12-desaturase, ∆6-desaturase, ∆5-desaturase, ∆5-elongase and/or ∆6-elongase gene. The scope of the invention is to comprise each and all of these nucleotide variation(s) and resulting amino acid polymorphisms in the ∆12-desaturase, ∆6-desaturase, ∆5-desaturase, ∆5-elongase and/or ∆6-elongase which are the result of natural variation and which do not essentially modify the enzymatic activity.
Essential enzymatic activity of the ∆12-desaturase, ∆6-desaturase, ∆6-elongase, ∆5-elongase or ∆5-desaturase used in the process according to the invention is understood as meaning that they retain an enzymatic activity of at least 10%, preferably of at least 20%, especially preferably of at least 30%, 40%, 50% or at least 60% and most preferably at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% in comparison with the proteins/enzymes encoded by the sequence and its derivatives and that they are thus capable of participating in the metabolism of compounds which are required for the synthesis of fatty acids, fatty acid esters such as diacylglycerides and/or triacylglycerides in a plant or plant cell or in the transport of molecules across membranes, meaning C18-, C20- or C14-carbon chains in the fatty acid molecule with double bonds at at least two, advantageously three, four or five, positions.
Likewise, the scope of the invention comprises nucleic acid molecules which hybridize under stringent conditions with the complementary strand of the ∆12-desaturase, ∆6-desaturase, ∆5-desaturase, ∆5-elongase and/or ∆6-elongase nucleic acids used. The term "hybridizes under stringent conditions" as used in the present context is to describe hybridization and washing conditions under which nucleotide sequences with at least 60% homology to one another usually remain hybridized with one another. Conditions are preferably such that sequences with at least approximately 65%, 70%, 80% or 90%, preferably at least approximately 91%, 92%, 93%, 94% or 95%, and especially preferably at least approximately 96%, 97%, 98%, 99% or more homology to one another usually remain hybridized to one another. These stringent conditions are

known to the skilled worker and described, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6.
A preferred, nonlimiting, example of stringent hybridization conditions is hybridizations in 6 x sodium chloride/sodium citrate (= SSC) at approximately 45°C, followed by one or more washing steps in 0.2 x SSC, 0.1% SDS at 50 to 65°C. The skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example when organic solvents are present, regarding temperature and buffer concentration. Under "standard hybridization conditions", for example, the hybridization temperature is, depending on the type of nucleic acid, between 42°C and 58°C in aqueous buffer with a concentration of 0.1 to 5 x SSC (pH 7.2). If organic solvents, for example 50% formamide, are present in the abovementioned buffer, the temperature under standard conditions is approximately 42°C. Preferably the hybridization conditions for DNA:DNA hybrids, for example, are 0.1 x SSC and 20°C to 45°C, preferably 30°C to 45°C. Preferably the hybridization conditions for DNA:RNA hybrids are, for example, 0.1 x SSC and 30°C to 55°C, preferably 45°C to 55°C. The abovementioned hybridization temperatures are determined for a nucleic acid with approximately 100 bp (= base pairs) in length and with a G + C content of 50% in the absence of formamide. The skilled worker knows how to determine the required hybridization conditions on the basis of textbooks such as Sambrook et a(., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Eds.) 1985, "Nucleic Acids Hybridization: A Practical Approach", IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991, "Essential Molecular Biology: A Practical Approach", IRL Press at Oxford University Press, Oxford.
By introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence, it is possible to generate an isolated nucleic acid molecule which encodes a ∆12-desaturase, ∆6-desaturase, ∆5-desaturase, ∆5-elongase and/or ∆6-elongase with one or more amino acid substitutions, additions or deletions. Mutations can be introduced into one of the sequences by means of standard techniques, such as site-specific mutagenesis and PCR-mediated mutagenesis. It is preferred to generate conservative amino acid substitutions in one or more of the above nonessential amino acid radicals. In a "conservative amino acid substitution", the amino acid radical is replaced by an amino acid radical with a similar side chain. Families of amino acid radicals with similar side chains have been defined in the art. These families comprise amino acids with basic side chains (for example lysine, arginine, histidine), acidic side chains (for example aspartic acid, glutamic acid), uncharged polar side chains (for example glycine, asparagine, giutamine, serine, threonine, tyrosine,

cysteine), unpolarside chains (for example alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (for example threonine, valine, isoleucine) and aromatic side chains (for example tyrosine, phenylalanine, tryptophan, histidine). A predicted nonessential amino acid radical in a ∆12-desaturase, ∆6-desaturase, ∆5-desaturase, ∆5-elongase or ∆6-elongase is thus preferably replaced by another amino acid radical from the same family of side chains. In another embodiment, the mutations can, alternatively, be introduced randoµly over all or part of the sequence encoding the ∆12-desaturase, ∆6-desaturase, ∆5-desaturase, ∆5-elongase or ∆6-elongase, for example by saturation mutagenesis, and the resulting mutants can be screened by recombinant expression for the hereindescribed ∆12-desaturase, ∆6-desaturase, ∆5-desaturase, ∆5-elongase or ∆6-elongase activity in order to identify mutants which have retained the ∆12-desaturase, ∆6-desaturase, ∆5-desaturase, ∆5-elongase or ∆6-elongase activity.
The polyunsaturated fatty acids produced in the process according to the invention advantageously comprise at least two, preferably three, four, five or six, double bonds. The fatty acids especially preferably comprise four, five or six double bonds. Fatty acids produced in the process preferably have a length of 20 C or 22 C atoms.
Saturated fatty acids are preferably reacted to a minor degree with the nucleic acids used in the process, or not at all. "A minor degree" is understood as meaning that, in comparison with polyunsaturated fatty acids, the saturated fatty acids are reacted with less than 5%, preferably with less than 3%, especially preferably with less than 2%, most preferably with less than 1, 0.5, 0.25 or 0.125% of the activity. The fatty acids produced may constitute the only product of the process or else may be present in a fatty acid mixture.
The polyunsaturated fatty acids produced in the process are advantageously bound in membrane liµl ds and/or triacylglycerides, but may also occur in the organisms as free fatty acids or else bound in the form of other fatty acid esters. In this context, they may be present as "pure products" or else advantageously in the form of mixtures of various fatty acids or mixtures of different glycerides. The various fatty acids which are bound in the triacylglycerides can be derived from short-chain fatty acids with 4 to 6 C atoms, medium-chain fatty acids with 8 to 12 C atoms or long-chain fatty acids with 14 to 24 C atoms, preferred are the long-chain fatty adds, especially preferred are the long-chain fatty acids LCPUFAs of C18-, C20- and/or C14-fatty acids, very especially preferred are the long-chain fatty acids LCPUFAs of C2
The process according to the invention advantageously yields fatty acid esters with polyunsaturated C18-, C20- and/or C22-fatty acid molecules with at least two double bonds in the fatty acid ester, advantageously with at least three, four, five or six double bonds in the fatty acid ester, especially advantageously four, five or six double bonds in the fatty acid ester, very especially advantageously at least five or six double bonds in the fatty acid ester. This advantageously leads to the synthesis of linoleic acid (=LA, C18:2A9'12), Y-Knolenic add (= GLA, C18:3∆6'9'12), stearidonic acid (= SDA, C18:4∆6'9'12-15), dihomo-Y-linolenic acid (= DGLA, 20:3A8'11'14), ω3--eicosatetraenoic acid (= ETA, C20:4∆5'8'11'14), arachidonic acid (ARA, C20:4∆5'8'11'14), eicosapentaenoic acid (EPA, C20:4∆58111'14) or mixtures of these, ω3-eicosapentaenoic acid (= ETA, C20:4∆5t81ii,i4,i7)j arachjdonic acid (ARA, C20:4∆5'8'11'14), eicosapentaenoic acid (EPA, C20:5∆5,8,ii1i4,i7)j us-docosapentaenoic acid (C22:5∆4'710'13-16), ω6--docosapentaenoic acid (C22:4∆7i1°'13'16), ω3--docosapentaenoic acid (=DPA, C22:5∆7lia13'16'19), docosahexaenoic acid (= DHA, C22:6∆4i7>10'13/!6'19) or their mixtures are preferably produced, and ARA, EPA and/or DHA are very especially produced. ω3-Fatty acids such as EPA and/or DHA, preferably DHA, are advantageously produced.
The fatty acid esters with polyunsaturated C18-, C20- and/or C22-fatty acid molecules, advantageously with polyunsaturated C2cr and/or C22-fatty acid molecules, can be isolated in the form of an oil or liµl d, for example in the form of compounds such as sphingoliµl ds, phosphoglycerides, liµl ds, glycoliµl ds such as glycosphingoliµl ds, phospholiµl ds such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol, monoacylglycerides, diacylglycerides, triacylglycerides or other fatty acid esters such as the acetyl-coenzyme A esters which comprise the polyunsaturated fatty acids with at least two, three, four, five or six, preferably four, five or six, especially preferably five or six, double bonds, from the plants which were used for the preparation of the fatty acid esters. Preferably, they are isolated in the form of their diacylglycerides, triacylglycerides and/or in the form of phosphatidylcholine, especially preferably in the form of the triacylglycerides. In addition to these esters, the polyunsaturated fatty acids are also present in the plants as free fatty acids or bound in other compounds. As a rule, the various abovementioned compounds (fatty acid esters and free fatty acids) are present in the organisms with an approximate distribution of 80 to 90% by weight of triglycerides, 2 to 5% by weight of diglycerides, 5 to 10% by weight of monoglycerides, 1 to 5% by weight of free fatty acids, 2 to 8% by weight of phospholiµl ds, the total of the various compounds amounting to 100% by weight.

In the method(s) according to the invention (for the purposes of the invention and the disclosure shown herein, the singular is to comprise the plural and vice versa), the LCPUFAs produced are produced in a content of at least 3, 5, 6, 7 or 8% by weight, advantageously at least 9, 10, 11, 12, 13, 14 or 15% by weight, preferably at least 16, 17, 18, 19 or 20% by weight, especially preferably at least 21, 22, 23, 24 or 25% by weight, very especially preferably at least 26, 27, 28, 29 or 30% by weight based on the total fatty acids in the transgenic organisms, advantageously in the seeds of the transgenic plants. Here, C18- and/or C20-fatty acids which are present in the host organisms are advantageously converted into the corresponding products such as ARA, EPA, DPA or DHA, to mention but a few by way of example, at the rate of at least 10%, advantageously at least 20%, especially advantageously at least 30%, very especially advantageously at least 40%. The fatty acids are advantageously produced in bound form.
Polyunsaturated C20-fatty acids with four or five double bonds in the molecule are advantageously produced in the process in a content of all such fatty acids together of at least 15, 16, 17, 18, 19, or 20% by weight, advantageously at least 21, 22, 23, 24 or 25% by weight, especially advantageously at least 26, 27, 28, 29 or 30% by weight based on the total fatty acids in the seeds of the transgenic plants.
Poiyunsaturated C20- and/or C22-fatty acids with four, five or six double bonds in the molecule are advantageously produced in the process in a content of all such fatty acids together of at least 15, 16, 17, 18, 19, or 20% by weight, advantageously at least
21, 22, 23, 24 or 25% by weight, especially advantageously at least 26, 27, 28, 29 or 30% by weight, very especially advantageously at least 31, 32, 33, 34 or 35% by weight based on the total fatty acids in the seeds of the transgenic plants.
ARA is produced in the process according to the invention in a content of at least 3, 5, 6, 7, 8, 9 or 10% by weight, advantageously at least 11, 12, 13, 14 or 15% by weight, preferably at least 16, 17, 18, 19 or 20% by weight, especially preferably at least 21,
22, 23, 24 or 25% by weight, most preferably at least 26% by weight, based on the total liµl d content in the seeds of the transgenic plants.
EPA is produced in the process according to the invention in a content of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1% by weight, advantageously at least 2, 3, 4 or 5% by weight, preferably at least 6, 7, 8, 9 or 10% by weight, especially preferably at least 11, 12, 13, 14 or 15% by weight and most preferably at least 16% by weight, based on the total liµl d content in the seeds of transgenic plants.

DHA is produced in the process according to the invention in a content of at least 0.01 or 0.02% by weight, advantageously at least 0.03 or 0.05% by weight, advantageously at least 0.09 or 0.1% by weight, especially preferably at least 0.2 or 0.3% by weight and most preferably at least 0.35% by weight, based on the total liµl d content in the seeds of the transgenic plants.
It is possible, with the aid of the nucleic acids used in the process according to the invention, for these unsaturated fatty acids to be positioned at the sn1, sn2 and/or sn3 position of the triglycerides which have advantageously been produced. Since in the process according to the invention the starting compounds linoleic acid (C18:2) and linolenic acid (C18:3) pass through a plurality of reaction steps, the end product of the process, such as, for example, arachidonic acid (ARA), eicosapentaenoic acid (EPA), oj6-docosapentaenoic acid or DHA, are not obtained as absolutely pure products, small traces of the precursors are also always present in the end product. If, for example, both linoleic acid and linolenic acid are present in the starting organism, or the starting plants, the end product, such as ARA, EPA or DHA, are present as mixtures. It is advantageous that, in the end product ARA or DHA, only minor amounts of the in each case other end product should be present. This is why, in a DHA-comprising liµl d and/or oil, less than 15, 14, 13, 12 or 11% by weight, advantageously less than 10, 9, 8, 7, 6 or 5% by weight, especially advantageously less than 4, 3, 2 or 1% by weight, of EPA and/or ARA should be present. This is why, in a EPA-comprising liµl d and/or oil, less than 15, 14, 13, 12 or 11% by weight, advantageously less than 10, 9, 8, 7, 6 or 5% by weight, especially advantageously less than 4, 3, 2 or 1% by weight, of ARA should be present. This is also why less than 15, 14, 13, 12 or 11% by weight, advantageously less than 10, 9, 8, 7, 6 or 5% by weight, especially advantageously less than 4, 3, 2 or 1% by weight of EPA and/or DHA should be present in an ARA-comprising liµl d and/or oil.
However, mixtures of different polyunsaturated C20- and/or C22-fatty acids in one product may also be desirable. In such cases, DHA-comprising liµl ds and/or oils may comprise at least 1, 2, 3, 4 or 5% by weight of ARA and/or EPA, advantageously at least 6, 7 or 8% by weight, especially advantageously at least 9, 10,11, 12, 13, 14 or 15% by weight, very especially advantageously at least 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25% by weight, based on the total liµl d content in the seeds of the transgenic plants.

The precursors should advantageously not amount to more than 20% by weight, preferably not to more than 15% by weight, especially preferably not to more than 10% by weight, very especially preferably not to more than 5% by weight, based on the amount of the end product in question. Advantageously, only ARA, EPA or only DHA, bound or as free acids, are produced as end products in the process of the invention in a transgenic plant. If the compounds ARA, EPA and DHA are produced simultaneously, they are advantageously produced in a ratio of at least 1:1:2 (EPA:ARA:DHA), advantageously at least 1:1:3, preferably 1:1:4, especially preferably 1:1:5. If the compounds ARA and EPA are produced simultaneously, they are advantageously produced, in the plant, in a ratio of at least 1:6 (EPA:ARA), advantageously of at least 1:8, preferably of at least 1:10, especially preferably of at least 1:12.
Fatty acid esters or fatty acid mixtures produced by the process according to the invention advantageously comprise 6 to 15% of pal.,itic acid, 1 to 6% of stearic acid, 7-85% of oleic acid, 0.5 to 8% of vaccenic acid, 0.1 to 1 % of arachic acid, 7 to 25% of saturated fatty acids, 8 to 85% of monounsaturated fatty acids and 60 to 85% of polyunsaturated fatty acids, in each case based on 100% and on the total fatty add content of the organisms.
Moreover, the fatty acid esters or fatty acid mixtures which have been produced by the process of the invention advantageously comprise fatty acids selected from the group of the fatty acids erucic acid (13-docosaenoic acid), sterculic acid (9,10-methyleneoctadec-9-enoic acid), malvalic acid (8,9-methyleneheptadec-8-enoic acid, chaulmoogric acid (cyclopentenedodecanoic acid), furan fatty acid (9,12-epoxyoctadeca-9,11-dienoic acid), vernoiic acid (9,10-epoxyoctadec-12-enoic acid), tariric acid (6-octadecynoic acid), 6-nonadecynoic acid, santalbic acid (t11-octadecen-9~ynoic acid), 6,9-octadecenynoic acid, pyrulic acid (t10-heptadecen-8-ynoic acid), crepenyninic acid (9-octadecen-12-ynoic acid), 13,14-dihydrooropheic acid, octadecen-13-ene-9,11-diynoic acid, petroselenic acid (cis-6-octadecenoic acid), 9c,12t-octadecadienoic acid, calendulic acid (8t10t12c-octadecatrienoic acid), catalµl c acid (9t11t13c-octadecatrienoic acid), eleostearic acid (9c11t13t-octadecatrienoic acid), jacaric acid (8c10t12c-octadecatrienoic acid), punicic acid (9c11t13c-octadecatrienoic acid), parinaric acid (9c11t13t15c-octadecatetraenoic acid), µl nolenic acid (all-cis-5,9,12-octadecatrienoic acid), laballenic acid (5,6-octadecadienallenic acid), ricinoleic acid (12-hydroxyoleic acid) and/or coriolic acid (13-hydroxy-9c,11t-octadecadienoic acid). The abovementioned fatty acids are, as a rule, advantageously only found in traces in the fatty acid esters or fatty acid mixtures produced by the process according

to the invention, that is to say that, based on the total fatty acids, they occur to less than 30%, preferably to less than 25%, 24%, 23%, 22% or 21%, especially preferably to less than 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5%, very especially preferably to less than 4%, 3%, 2% or 1%. In a further preferred form of the invention, these abovementioned fatty acids occur to less than 0.9%, 0.8%, 0.7%, 0.6% or 0.5%, especially preferably to less than 0.4%, 0.3%, 0.2%, 0.1%, based on the total fatty acids. The fatty acid esters or fatty acid mixtures produced by the process according to the invention advantageously comprise less than 0.1%, based on the total fatty acids, or no butyric acid, no cholesterol, no clupanodonic acid (= docosapentaenoic acid, C22:5∆4'8'12'15'21) and no nisinic acid (tetracosahexaenoic acid, C23:6A3'8'1215-18'21).
Owing to the nucleic acid sequences according to the invention or nucleic acid sequences used in the process according to the invention, an increase in the yield of polyunsaturated fatty acids, mainly ARA and EPA, but also DHA, of at least 50, 80 or 100%, advantageously at least 150, 200 or 250%, especially advantageously at least 300, 400, 500, 600, 700, 800 or 900%, very especially advantageously at least 1000, 1100, 1200, 1300, 1400 or 1500% in comparison with the nontransgenic starting plant, for example a plant such as Brassica juncea, Brassica napus, Camelina sativa, Arabidopsis thanliana or Linum usitatissimum when compared by means of GC analysis; see Examples.
Advantageously, as described above, the polyunsaturated C20- and/or C22-fatty acids with four, five or six double bonds in the molecule, which are produced in the process, will comprise in the seeds of plants which comprise only very small amounts of C12:0-or C14:0-fatty acids, or none at all. Even shorter saturated fatty acids, such as the fatty acids C4:0, C6:0, C8:0 or C10:0 should not be present in the liµl d and/or oil or only in very small amounts. Only very small amounts are advantageously understood as amounts which, in GC analysis, are advantageously under 5, 4, 3, 2 or 1%, advantageously under 0.9, 0.8, 0.7, 0.6 or 0.5%, especially advantageously under 0.4, 0.3, 0.2 or 0.1%, very especially preferably under 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.01 units area in the GC. The fatty acid C16:0 should advantageously be in a range of from 1 to 28% GC units area. The fatty acid C16:0 should advantageously be present in GC units area in amounts of less than 25%, 20%, 15% or 10%, advantageously less than 9%, 8%, 7%, 6% or 5%, especially advantageously less than 4%, 3%, 2% or 1% or not at all, in the liµl ds, oils and/or free fatty acids. The fatty acid C16:1 should advantageously amount to less than 1, 0.5, 0.4, 0.3, 0.2 or 0.1%, especially advantageously 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.01 units area in the GC. Very especially preferably, the fatty acid C16:1 should not be present in

the oils and/or liµl ds produced by the process. The same applies to the fatty acids 015:0, 017:0, C16:1A3trans, C16:4M-710'13 and C18:5A3'6-9'12'15. Besides oleic acid (C18:1A9)( the isomers (C18:1∆7, 18:1A11) may also be present in the liµl ds, oils or free fatty acids. Advantageously in amounts of less than 5%, 4%, 3%, 2% or 1%, measured as units GO area. The fatty acids C20:0, C20:1, C24:0 and C24:1 should in each case be in the range of from 0 to 1%, 0 to 3% and 0 to 5%, respectively, units GC area. Furthermore, little dihomo-y-linolenic acid (= DGLA) should be detectable in the GC analysis in units GC area in the seed oil and/or seed liµl d. Little is understood as meaning less than 2, 1.9, 1.8, 1.7, 1.6 or 1.5%, advantageously less than 1.4, 1.3, 1.2, 1.1 or 1%, especially advantageously less than 0.9, 0.8, 0.7, 0.6, 0.5 or 0.4% in units GC area.
In a preferred embodiment of the process, DGLA and ARA should be produced in a ratio of from 1:1 up to 1:100, advantageously from 1:2 up to 1:80, especially advantageously from 1:3 up to 1:70, very especially from 1:5 up to 1:60.
In a further preferred embodiment, DGLA and EPA should be produced in a ratio of from 1:1 up to 1:100, advantageously from 1:2 up to 1:80, especially advantageously from 1:3 up to 1:70, very especially from 1:5 up to 1:60.
The liµl ds and/or oils produced in the process according to the invention should advantageously have a high unsaturated, advantgeously polyunsaturated, fatty acid content of at least 30, 40 or 50% by weight, advantageously at least 60, 70 or 80% by weight, based on the total fatty acid content in the seeds of the transgenic plants.
All saturated fatty acids together should advantageously only amount to a small quantity in the plants preferably used in the process according to the invention. In this context, a small amount is understood as meaning an amount of less than 15%, 14%, 13%, 12%, 11% or 10%, preferably less than 9%, 8%, 7% or 6%, in units GC area.
Furthermore, the genes for the synthesis of the polyunsaturated fatty acids, which are used in the process and which have been introduced, in the process, via different processes, advantageously as host plant, should advantageously have a higher oil content than protein content in the seed, advantageous plants have an oil/protein content ratio of from 5:1, 4:1, 3:1, 2:1 or 1:1. In this context, the oil content based on the total weight of the seed should be in a range of 15-55%, advantageously between 25-50%, especially advantageously between 35-50%. Advantageous host plants used in the process should have a distribution of the unsaturated fatty acids such as oleic

acid, linoleic acid and linolenic acid, which are the starting compounds in the process according to the invention for the synthesis of polyunsaturated fatty acids, in the sn1, sn2 and sn3 position of the triglyceride, as shown in Table 5 hereinbelow, where rows No. 1-7 represent different advantageous alternatives of such distributions, n.p. means not present.
The rows show the ratios of the following plants: row 1 = Arachis hypogaea, row 2 = Brassica napus, row 3 = Glycine max, row 4 = Linum usitatissimum, row 5 = Zea mays, row 6 = Olea europaea and row 7 = Theobroma cacao.
Host plants which are advantageous for the process are those which have a high oleic acid content, that means at least 40, 50, 60 or 70% by weight based on the total fatty acid content of the plant, in comparison with linoleic acid and/or linolenic acid in the liµl ds and/or oils, especially in the triglyceride, such as, for example, Anarcardium occidental, Argania sµl nosa, Bombax malabaricum, Brassica napus, Butyrospermum parkii, high-oleic safflower (Carthamus tinctorius), Citrullus colocythis, Corylus avellana, Curcurbita foetidissima, Curcurbita pepo, Guizotia abyssinica, high-oleic sunflower (Helianthus annus), Macadamia intergrifolia, Nigella sativa, Olea europaea, Papaversomniferium, Passiflora edulis, Persea americana, Prunus amygdalis, Prunus armeniaca, Prunus dulcis, Prunus communis, Sesamum indicum, Simarouba glauca, Thea sasumgua, or Theobroma cacao. Further advantageous plants have a higher content of the unsaturated fatty acids oleic acid, linoleic acid and a-linolenic acid in the sn2 position in comparison with the other positions sn1 and sn3. A higher content is understood as meaning ratios of (sn1:sn2:sn3) 1:1.1:1, 1:1.5:1 to 1:3:1. Advantageous

plants such as Actinidia chinensis, Aleurites moluccana, Arnebia griffithii, Brassica alba, Brassica hirta, Brassica nigra, Brassica juncea, Brassica carinata, Camelina sativa, Cannabis sativa, Echium rubrum, Echium vulgare, Humulus lupulus, Juglans regia, Linum usitatissimum, Ocimum spp., Perilla frutescens, Portulaca oleracea, Prunus cerasus, Salicornia bigelovii, Salvia hispanica are also those which have a high a-Iinolenic acid content in the liµl d and/or oil of the plant, that is to say an a-linolenic acid content of at least 10, 15 or 20% by weight, advantageously at least 25, 30, 35, 40, 45 or 50% by weight, based on the total fatty acid content of the plant. Very especially advantageous plants likewise show an advantageous preference for the sn2 position over the positions sn1 and sn3 in the triglyceride of from 1:1.1:1, 1:1.5:1 to 1:3:1 for the arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid produced in the process.
Plants used for the process should advantageously have an erucic acid content of less than 2% by weight based on the total fatty acid content of the plant. Also, the content of saturated fatty acids C16:0 and/or C18:0 should advantageously be less than 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10% by weight, advantageously less than 9, 8, 7, 6 or 5% by weight, based on the total fatty acid content of the plant. Also, longer fatty acids such as C20:0 or C22:1 should advantageously not be present, or only in small amounts, advantageously in amounts of less than 4, 3, 2 or 1% by weight, advantageously less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1% by weight based on the total fatty acid content of the plant in the plants used in the process. Tyµl cally, C16:1 is not present as fatty acid, or only present in small amounts, in the plants used for the process according to the invention. Small amounts are advantageously understood as meaning fatty acid contents which are less than 4, 3, 2 or 1% by weight, advantageously less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1% by weight based on the total fatty acid content of the plant.
For economic reasons, that is to say because of the area under cultivation and the oil yield, plants which are grown on a large scale, such as soybean, oilseed rape, mustard, Camelina, linseed, sunflower, oil pal.,, cotton, sesame, maize, olive, are preferred, preferably oilseed rape, Camelina, linseed, sunflower are used frequently as host plant in the process.
Chemically pure polyunsaturated fatty acids or fatty acid compositions can also be synthesized by the processes described above. To this end, the fatty acids or the fatty acid compositions are isolated from the plants, advantageously the seeds of the plants, in the known manner, for example via crushing the seeds, such as grinding, followed

Lycopersicon pyriforme, Solanum integrifolium or Solanum lycopersicum [tomato], Sterculiaceae, such as the genus Theobroma, for example the genus and species Theobroma cacao [cacao] or Theaceae, such as the genus Camellia, for example the genus and species Camellia sinensis [tea]. Further plants which may be mentioned are the genus and species Argania sµl nosa, Arnebia griffithii, Adansonia digitata, Orbignya martiana, Carum carvi, Bertholletia excelsa, Aleurites moiuccana, Hydnocarpus kursii, Salvia hispanica, Vitis vinifera, Corvlus avellana, Humulus lupus, Hyptis sµl cigera and Shorea stenoptera.
Plants which are advantageously used in the process according to the invention are transgenic plants such as dicotyledonous or monocotyledonous plants. Plants which are especially advantageously used in the process according to the invention are transgenic plants which belong to the oil-producing plants, that is to say which are used for the production of oils, such as, preferably, oil fruit crops which comprise large amounts of liµl d compounds, such as peanut, oilseed rape, canola, sunflower, safflower (Carthamus tinctoria), poppy, mustard, hemp, castor-oil plant, olive, sesame, Calendula, Punica, evening primrose, mullein, thistle, wild roses, hazelnut, al.,ond, macadamia, avocado, bay, pumpkin/squash, linseed, soybean, µl stachios, borage, trees (oil pal.,, coconut, walnut) or crops such as maize, wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper, Tagetes, Solanaceae plants such as potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa or bushy plants (coffee, cacao, tea), Salix species, and perrenial grasses and fodder crops.
Preferred plants according to the invention are oilseed and oil crop plants such as peanut, oilseed rape, canola, sunflower, safflower, poppy, Indian mustard, mustard, hemp, castor-oil plant, olive, Calendula, Punica, evening primrose, pumpkin/squash, linseed, soybean, borage, trees (oil pal.,, coconut). Especially preferred are plants which are high in C18:2- and/or C18:3-fatty acids, such as sunflower, safflower, tobacco, mullein, sesame, cotton, pumpkin/squash, poppy, evening primrose, walnut, linseed, hemp, thistle or safflower. Very especially preferred plants are plants such as safflower, sunflower, poppy, evening primrose, walnut, linseed, Indian mustard, Camelina or hemp.
It is advantageous for the above-described processes according to the invention to additionally introduce, into the plant, further nucleic acids which encode enzymes of the fatty acid or liµl d metabolism, in addition to the nucleic acids introduced in steps (a) to (e) or (a) to (c) of the process, and the optionally introduced nucleic acid sequences which encode the oo3-desaturases and/or the ∆12-desaturases.

In principle, all genes of the fatty acid or liµl cl metabolism can be used in the process for the production of polyunsaturated fatty acids, advantageously in combination with the ∆5-elongase(s), ∆6-elongase(s) and/or ω3-desaturases [for the purposes of the present invention, the plural is understood as encompassing the singular and vice versa]. Genes of the fatty acid or (iµl d metabolism selected from the group consisting of acyl-CoA dehydrogenase(s), acyl-ACP [= acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyl transferase(s), acyl-CoA:lysophospholiµl d acyltransferases, fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allenoxide synthases, hydroperoxide lyases or fatty acid elongase(s) are advantageously used in combination with the ∆5-elongase, ∆6-elongase and/or ω3-desaturase. Genes selected from the group of the ∆4-desaturases, ∆5-desaturases, ∆6-desaturases, ∆8-desaturases, ∆9-desaturases, ∆12-desaturases, ∆6-elongases or ∆9-elongases are especially preferably used in combination with the above genes for the ∆5-elongase, ∆6-elongase and/or ω3-desaturase, it being possible to use individual genes or a plurality of genes in combination. The abovementioned genes are advantageously used in combination with the ∆6-elongase, ∆5-elongase, ∆5-desaturase, ∆6-desaturase and/or ∆12-desaturase used in accordance with the invention.
Genes selected from the group of the ∆8-desaturases, ∆9-desaturases, ∆5-elongase or ∆9-elongases are especially preferably used in combination with the abovementioned genes.
Owing to the enzymatic activity of the nucleic acids used in the process according to the invention which encode polypeptides with ∆6-elongase, ∆6-desaturase, ∆5-desaturase and/or ∆12-desaturase activity, advantageously in combination with nucleic acid sequences which encode polypeptides of the fatty acid or liµl d metabolism, such as polypeptides with A8~desaturase, or ∆5- or ∆9-elongase activity, a wide range of polyunsaturated fatty acids can be produced in the process according to the invention. Depending on the choice of plants used for the process according to the invention, mixtures of the various polyunsaturated fatty acids or individual polyunsaturated fatty acids, such as EPA or ARA, can be produced in free or bound form. Depending on the prevailing fatty acid composition in the starting plant (C18:2- or C18:3-fatty acids), fatty acids which are derived from C18;2-fatty acids, such as GLA, DGLA or ARA, or fatty acids which are derived from C18:3-fatty acids, such as SDA, ETA or EPA, are thus obtained. If only iinoleic acid (= LA, C18:2A9>12) is present as unsaturated fatty acid in

the plant used for the process, the process can only afford GLA, DGLA and ARA as products, all of which can be present as free fatty acids or in bound form. If only a-linolenic acid (= ALA, C18:3∆9i12t15) is present as unsaturated fatty acid in the'plant used for the process, as is the case, for example, in linseed, the process can only afford SDA, ETA or EPA as products, all of which can be present as free fatty acids or in bound form, as described above.
Owing to the activity of ∆6-desaturase and ∆6-elongase, products formed are, for example, GLA and DGLA, or SDA and ETA, respectively, depending, on the starting plant and the unsaturated fatty acid present therein. DGLA or ETA or mixtures of these are preferentially formed. If ∆5-desaturase is additionally introduced into the plant, ARA and/or EPA are also formed. If, moreover, genes which encode a ∆5-elongase and/or ∆4-desaturase activity are additionally introduced, the fatty acids DPA and/or DHA can be produced in the process according to the invention. Advantageously, only ARA, EPA and/or DHA or mixtures of these are synthesized, depending on the fatty acid present in the plant, which acts as starting substance for the synthesis. Since biosynthetic cascades are involved, the end products in question are not present in pure form in the organisms. Small amounts of the precursor compounds are always additionally present in the end product. These small amounts-amount to less than 20% by weight, advantageously less than 15% by weight, especially advantacjfeously less than 10% by weight, most advantageously less than 5, 4, 3, 2 or 1% by weight, based on the end products DGLA, ETA or their mixtures, or ARA, EPA or their mixtures, or ARA, EPA, DHA or their mixtures.
In addition to the production directly in the plant, of the starting fatty acids for the enzymes used in the process of the invention, the fatty acids can also be fed externally. The production in the plant is preferred for reasons of economy. Substrates which are preferred for the production of ARA are linoleic acid (C18:2A9'12), ylinolenic acid (C18:3∆6t9t12) and dihomo-y-linolenic acid (20:3 ABi11'14). Substrates which are preferred for the production of EPA are linolenic acid (C18:3∆912115), stearidonic acid (C18:4∆6>9'12'15) and eicosatetraenoic acid (C20:4A8'11iK17). Substrates which are preferred for the production of DHA are linolenic acid (C18:3∆9'12'15), stearidonic acid (C18:4∆6'9"12'15), eicosatetraenoic acid (C20:4A8i11i14'1?), EPA and DPA.
In comparison with the human elongases or elongases from non-human animals, such as those from Oncorhynchus, Xenopus or Ciona, the ∆5-elongases according to the invention have the advantageous characteristic that they do not elongate C22-fatty acids to the corresponding C24-fatty acids. Furthermore, they advantageously do not convert

fatty acids with a double bond in the ∆6-position, as is the case with the human elongases or the elongases from non-human animals. Especially advantageously ∆5-elongases preferentially only convert unsaturated C2£rfatty acids. These advantageous ∆5-elongases contain some putative transmembrane helices (5 - 7). Advantageously, only C20-fatty acids with one double bond in the ∆5-position are converted, with CJ3-C20-fatty acids being preferred (EPA). Moreover, in a preferred embodiment of the invention, they have the characteristic that, besides the ∆5-elongase activity, they advantageously have no, or only relatively low, ∆6-elongase activity. In contrast, the human elongases or non-human animal elongases have approximately the same activity towards fatty acids with a ∆6- or ∆5-double bond. These advantageous elongases are referred to what are known as monofunctional elongases. In contrast, the human elongases or the non-human animal elongases are referred to as multifunctional elongases, which, besides the abovementioned substrates, also convert monounsaturated C16- and C18-fatty acids, for example with ∆9- or A11-double bonds. In a yeast feeding text, in which EPA was added to the yeast as the substrate, the monofunctional elongases convert at least 15% by weight of the added EPA into docosapentaenoic acid (DPA, C22:5∆7'10'13'16'19), advantageously at least 20% by weight, especially advantageously at least 25% by weight. If y-linolenic acid (= GLA, C18:3∆6i9>12) is added as the substrate, this acid is advantageously not elongated at all. Likewise, C18:3∆69'12 is not elongated. In another advantageous embodiment, less than 60% by weight of the added GLA is converted into dihomo-v-linolenic acid (= C20:3 A8'11'14), advantageously less than 55% by weight, preferably less than 50% by weight, especially advantageously less than 45% by weight, very especially advantageously less than 40% by weight. In a further, very preferred embodiment of the ∆5-elongase activity according to the invention, GLA is not converted.
Figures 27 and 28 show the measured substrate specificities of the various elongases. Figure 27 shows the specificities of the multifunctional elongases from Xenopus laevis (Fig. 27 A), Ciona intestinalis (Fig. 27 B) and Oncorhynchus mykiss (Fig. 27 C). All these elongases convert a broad substrate spectrum. In the process according to the invention, this can lead to by-products, which must be converted by further enzymatic activities. This is why these enzymes are less preferred in the process according to the invention. The preferred monofunctional elongases and their substrate specificity are shown in Figure 28. Figure 28 A shows the specificity of the Ostreococcus tauri ∆5-elongase. This enzyme only converts fatty acids with a double bond in the ∆5-position. Advantageously, only C20-fatty acids are converted. A similarly high substrate specificity is shown by the Thallasiosira pseudonana ∆5-elongase (Fig. 28. C). Both the Ostreococcus tauri ∆6-elongase (Fig. 28 B) as that of Thallasiosira pseudonana (Fig.

28 D) advantageously only convert fatty acids with a double bond in the ∆6-position. Advantageously, only C18-fatty acids are converted. The ∆5-elongases from Arabidopsis thaliana and Euglena gracilis are also distinguished by their specificities.
Likewise, advantageous ∆6-elongases according to the invention are distinguished by a high specificity, that is to say that C18-fatty acids are preferentially elongated. They advantageously convert fatty acids with a double bond in the ∆6-position. Especially advantageous ∆6-elongases advantageously convert C18-fatty acids with three or four double bonds in the molecule, which fatty acids must comprise a double bond in the ∆6-position. Moreover, in a preferred embodiment of the invention, they have the characteristic that, besides the ∆6-elongase activity, they advantageously have no, or only relatively low, ∆5-elongase activity. In contrast, the human elongases or non-human animal elongases have approximately the same activity towards fatty acids with a ∆6- or ∆5-double bond. These advantageous elongases are referred to as what are known as monofunctionai elongases. In contrast, the human elongases or the non-human animal elongases are referred to as multifunctional elongases, which, besides the abovementioned substrates, also convert monounsaturated C16- and C18-fatty acids, for example with ∆9- or A11-double bonds. In a yeast feeding text, in which EPA has been added to the yeasts as the substrate, the monofunctionai elongases convert at least 10% by weight of the added a-linolenic acid (= ALA, C18:3∆9I2'15) or at least 40% by weight of added ylinolenic acid (= GLA, C18:3∆6,9,12), advantageously at least 20% by weight and 50% by weight, respectively, especially advantageously at least 25% by weight and 60% by weight, respectively. It is especially advantageous that C18:4∆6912115 (stearidonic acid) is also elongated. Here, SDA is converted to at least 40% by weight, advantageously to at least 50% by weight, especially advantageously to at least 60% by weight, very especially advantageously to at least 70% by weight. Especially advantageous ∆6-elongases show no, or only very low activity (less than 0.1% by weight conversion rate) toward the following substrates: C18:1∆6, C18:1A9, C18:1∆1\ C20:2∆11'14, C20:3∆11'K17, C20:3∆8'11'14, C20:4∆5-8'11-14, C20:5∆5'11'14'17 or C22'4∆7,|10,13',16
Figures 29 and 30 and Table 21 show the measured substrate specificities of the various elongases.
In comparison with the known oj3-desaturase, the ω3--desaturase used in the process according to the invention has the advantageous characteristic that it is capable of desaturating a broad spectrum of cu6-fatty adds, with C20- and C22-fatty acids such as C20:2-, C20:3-, C20:4-, C22.4' or C14s-fatty acids being preferentially desaturated. However,

the shorter C18-fatty acids such as C18:2- or C18;3-fatty acids are also advantageously desaturated. Owing to these characteristics of ω3-desaturase, it is advantageously possible to shift the fatty acid spectrum within an organism, advantageously within a plant or a fungus, from the ω6--fatty acids towards the ω3-fatty acids. The ω3-desaturase according to the invention preferentially desaturates C20-fatty acids. Within the organism, these fatty acids are converted to at least 10%, 15%, 20%, 25% or 30% from the existing fatty acid pool to give the corresponding ω3-fatty acids. In comparison with the C18-fatty acids, the activity of ω3-desaturase is lower by a factor of 10, that is to say only approximately 1.5 to 3% of the fatty acids present in the fatty acid pool are converted into the corresponding ω3-fatty acids. Preferred substrates of the ω3-desaturase according to the invention are the co6-fatty acids bound in phospholiµl ds. With reference to the desaturation of dihomo-Y-linolenic acid [C20:4∆8t1114], Figure 19 shows clearly that ω3-desaturase advantageously does not differentiate between fatty acids bound at the sn1 or sn2 position when desaturation takes place. Both fatty acids bound at the sn1 position and fatty acids bound in the sn2 position in the phospholiµl ds are desaturated. Another advantage is that iu3-desaturase converts a broad range of phospholiµl ds such as phosphatidylcholine (= PC), phosphatidylinositol (= µL S) or phosphatidylethanolamine (= PE). Finally, desaturation products are also found in the neutral liµl ds (= NL), i.e. in the triglycerides.
In comparison with the known ∆4-desatu rases, ∆5-desaturases and ∆6-desaturases, the advantage of the ∆4-desaturases, ∆5-desaturases and ∆6-desaturases used in the process according to the invention is that they can convert fatty acids which are bound to phospholiµl ds or CoA-fatty acid esters, advantageously CoA-fatty acid esters.
The ∆12-desaturases used in the process according to the invention advantageously convert oleic add (C18:1∆9) into linoleic acid (C18:2∆912) or C18:2∆6'9 into C18:3∆6'9'12 (= GLA). The ∆12-desaturases used advantageously convert fatty acids which are bound to phospholiµl ds or CoA-fatty acid esters, advantageously those which are bound to CoA-fatty acid esters.
Owing to the enzymatic activity of the nucleic acids used in the process according to the invention which encode polypeptides with ∆5-elongase, ∆6-elongase and/or co3» desaturase activity, advantageously in combination with nucleic acid sequences which encode polypeptides of the fatty acid or liµl d metabolism, such as additionally polypeptides with ∆4-, ∆5-, ∆6-, ∆8-, ∆12-desaturase or ∆5-, ∆6- or ∆9-elongase activity, a very wide range of polyunsaturated fatty acids can be produced in the process according to the invention. Depending on the choice of the advantageous

plants used for the process according to the invention, mixtures of the various poiyunsaturated fatty acids or individual polyunsaturated fatty acids such as EPA, ARA or DHA, can be produced in free or bound form. Depending on the prevailing fatty acid composition in the starting plant (C18:2- or C18:3-fatty acids), fatty acids which are derived from C18:2-fatty acids, such as GLA, DGLA or ARA, or which are derived from C18:3-fatty acids, such as SDA, ETA, EPA or DHA, are thus obtained. If only linoleic acid (= LA, C18:2∆912) is present as unsaturated fatty acid in the plant used for the process, the process can only afford GLA, DGLA and ARA as products, all of which can be present as free fatty adds or in bound form. By expressing the additional ω3-desaturase in plants, the fatty acid spectrum can be shifted towards a-linolenic acid, DPA and DHA. However, this shift in the fatty acid spectrum is only relatively limited. More advantageous is such a shift in plants which, as described hereinbelow, already have a high a-linolenic acid content. If only a-linolenic acid (= ALA, C18:3∆9'12'15) is present as unsaturated fatty acid in the plant, as is the case, for example, in linseed, the process can only afford SDA, ETA, EPA and/or DHA, which, as described above, may be present as free fatty acids or in bound form. Owing to the modification of the activity of the enzyme ∆5-elongase which plays a role in the synthesis, advantageously in combination with ∆4-, ∆5-, ∆6-, ∆12-desaturase and/or ∆6-elongase, or ∆4-, ∆5-, ∆8-, ∆12-desaturase, and/or ∆9-elongase, it is possible to produce, in a targeted fashion, only individual products in the abovementioned plants. Owing to the activity of ∆6-desaturase and ∆6-eiongase, for example, GLA and DGLA, or SDA and ETA, are formed, depending on the starting plant and unsaturated fatty acids. DGLA or ETA or mixtures of these are preferentially formed. If ∆5-desaturase, ∆5-elongase and ∆4-desaturase are additionally introduced into the organisms, advantageously into the plant, ARA, EPA and/or DHA are additionally formed. This also applies to organisms into which ∆8-desaturase and ∆9-elongase have previously been introduced. Advantageously, only ARA, EPA or DHA or their mixtures are synthesized, depending on the fatty acid present in the plant, which acts as starting substance for the synthesis. Since biosynthetic cascades are involved, the end products in question are not present in pure form in the organisms. Small amounts of the precursor compounds are always additionally present in the end product. These small amounts amount to less than 20% by weight, advantageously less than 15% by weight, especially advantageously less than 10% by weight, very especially advantageously less than 5, 4, 3, 2, or 1% by weight, based on the end product DGLA, ETA or their mixtures, or ARA, EPA, DHA or their mixtures, advantageously EPA or DHA or their mixtures.
The nucleic acid with the SEQ ID NO: 53, which is derived from trout and which can be used in the process according to the invention, encodes a protein with high specificity

for the two C18:4∆6'912'15" and C20:5∆5i8i11'14'17-fatty acids, which are precursors for the synthesis of DHA (precursors and synthesis of DHA, see Figure 1). However, other fatty acids too are elongated by the enzyme. The protein encoded by SEQ ID NO; 53 thus has specificity for ∆6- and ∆5-fatty acids with additionally one ω3--double bond (Figure 2). ∆5-elongase has a keto-acyl-CoA synthase activity which advantageously elongates fatty acid residues of acyl-CoA esters by 2 carbon atoms.
The synthesis of DHA in yeast (Saccharomyces cerevisiae) was detected by the gene product of the abovementioned fish ∆5-elongase gene and further ∆5-elongases, the ∆5-desaturase from Phaeodactylum and the ∆4-desaturase from Euglena (Figure 3).
In addition to the production directly in the transgenic organism, advantageously in the transgenic plant, of the starting fatty acids for the ∆5-elongases, ∆6-elongases, ∆9-elongases, ∆4-desaturases, ∆5-desaturases, ∆6-desaturases, ∆12-desaturases and/or ω3-desaturases advantageously used in the process according to the invention, the fatty acids can also be shed externally. The production in the organism is preferred for reasons of economy. Preferred substrates of ω3-desaturase are linoleic acid (C18:2∆912), v-linolenic acid (C18:3∆6'9>12), eicosadienoic acid (C20:2∆11-14), dihomo-y-linolenic acid (C20:3∆8n14), arachidonic acid (C20:4∆5'8'11-14), docosatetraenoic acid (C22:4∆7-10'13'16) and docosapentaenoic acid (C22:5∆4'7'10'13'15).
To increase the yield in the above-described process for the production of oils and/or triglycerides with an advantageously elevated content of polyunsaturated fatty acids, it is advantageous to increase the amount of starting product for the synthesis of fatty acids; this can be achieved for example by introducing, into the organism, a nucleic acid which encodes a polypeptide with ∆12-desaturase activity. This is particularly advantageous in oil-producing organisms such as those from the family of the Brassicaceae, such as the genus Brassica, for example oilseed rape; the family of the Elaeagnaceae, such as the genus Elaeagnus, for example the genus and species Olea europaea, or the family Fabaceae, such as the genus Glycine, for example the genus and species Glycine max, which are high in oleic acid. Since these organisms are only low in ftnoleic acid (Mikoklajczak et al., Journal of the American Oil Chemical Society, 38, 1961, 678 - 681), the use of the abovementioned ∆12-desaturases for producing the starting material linoleic acid is advantageous.
Nucleic acids used in the process according to the invention are advantageously derived from plants such as algae, for example algae of the family of the Prasinophyceae such as the genera Heteromastix, Mammella, Mantoniella, Micromonas, Nephroselmis, Ostreococcus, Prasinocladus, Prasinococcus, Pseudoscourfielda, Pycnococcus, Pyramimonas, Scherffelia or Tetraselmis such as the genera and species Heteromastix longifillis, Mamiella gilva, Mantoniella squamata, Micromonas pusilla, Nephroselmis olivacea, Nephroselmis pyriformis, Nephroselmis

rotunda, Ostreococcus tauri, Ostreococcus sp. Prasinocladus ascus, Prasinocladus lubricus, Pycnococcus provasolii, Pyramimonas amylifera, Pyramimonas disomata, Pyramimonas obovata, Pyramimonas orientalis, Pyramimonas parkeae, Pyramimonas sµl nifera, Pyramimonas sp., Tetraselmis aµl culata, Tetraselmis carteriaformis, Tetraselmis chui, Tetraselmis convolutae, Tetraselmis desikacharyi, Tetraselmis gracilis, Tetraselmis hazeni, Tetraseimis impellucida, Tetraselmis inconsµl cua, Tetraselmis levis, Tetraselmis maculata, Tetraselmis marina, Tetraselmis striata, Tetraselmis subcordiformis, Tetraselmis suecica, Tetraselmis tetrabrachia, Tetraselmis tetrathele, Tetraselmis verrucosa, Tetraselmis verrucosa fo. rubens or Tetraselmis sp. or from algae of the family Euglenaceae such as from the genera Ascoglena, Astasia) Colacium, Cyclidiopsis, Euglena, Euglenopsis, Hyalophacus, Khawkinea, Lepocindis, Phacus, Strombomonas or Trachelomonas such as the genera and species Euglena acus, Euglena geniculata, Euglena gracilis, Euglena mixocylindrica, Euglena rostrifera Euglena viridis, Colacium stentorium, Trachelomonas cylindrica or Trachelomonas volvocina. The nucleic acid sequences used in the process can also advantageously be derived from aigae, such as the alga Porphyridium cruentum, Isochrysis galbana or Chlorella minutissima, Chlorella vulgaris, Thraustochytrium aureum or Nannochloropsis oculata. The nucleic acids used are advantageously derived from algae of the genera Euglena, Mantoniella or Ostreococcus.
Further advantageous plants as sources for the nucleic acid sequences used in the process according to the invention are algae such as Isochrysis or Crypthecodinium, algae/diatoms such as Thalassiosira or Phaeodactylum, mosses such as Physcomitrella or Ceratodon, or higher plants such as the Primulaceae such as Aleuritia, Calendula stellata, Osteospermum sµl nescens or Osteospermum hyoseroides, microorganisms such as fungi, such as Aspergillus, Thraustochytrium, Phytophthora, Entomophthora, Mucor or Mortierella, bacteria such as Shewanella, yeasts or animals such as nematodes such as Caenorhabditis, insects, frogs, sea cucumber or fish. The isolated nucleic acid sequences according to the invention are advantageously derived from an animal of the order of the vertebrates. Preferably, the nucleic acid sequences are derived from the classes of the Vertebrata; Euteleostomi, Actinopterygii; Neopterygii; Teleostei; Euteleostei, Protacanthopterygii, Sal.,oniformes; Sal.,onidae or Oncorhynchus or Vertebrata, Amphibia, Anura, µl µl dae, Xenopus or Evertebrata such as Protochordata, Tunicata, Holothuroidea, Cionidae such as Amaroucium constellatum, Botryllus schlosseri, Ciona intestinalis, Molgula citrina, Molgula manhattensis, Perophora viridis or Styela partita. The nucleic acids are especially advantageously derived from fungi, animals, or from plants such as algae or mosses, preferably from the order of the Sal.,oniformes, such as the family of the Sal.,onidae, such as the genus Sal.,o, for example from the genera and species Oncorhynchus mykiss, Trutta trutta or Sal.,o trutta fario, from algae, such as the

genera Mantoniella or Ostreococcus, or from the diatoms such as the genera Thalassiosira or Phaeodactylum or from algae such as Crypthecodinium.
Advantageous nupleic acid used in the process according to the invention can also be i derived from microorganisms such as fungi such as the genus Mortierella, Phytium, for example the genus and species Mortierella alµl ina, Mortierelia elongata, Phytium irregulare, Phytium ultimum or bacteria such as the genus Shewanella, for example the genus and species Shewanella hanedai.
The process according to the invention advantageously employs the abovementioned nucleic acid sequences or their derivatives or homologs which encode polypeptides which retain the enzymatic activity of the proteins encoded by nucleic acid sequences. These sequences, individually or in combination with the nucleic acid sequences which encode ∆12-desaturase, ∆4-desaturase, ∆5-desaturase, ∆6-desaturase, ∆5-elongase, ∆6-elongase and/or ω3-desaturase, are cloned into expression constructs and used for the introduction into, and expression in, organisms. Owing to their construction, these expression constructs make possible an advantageous optimal synthesis of the polyunsaturated fatty acids produced in the process according to the invention.
In a preferred embodiment, the process furthermore comprises the step of obtaining a transgenic plant which comprises the nucleic acid sequences used in the process, where the plant is transformed with a nucleic acid sequence according to the invention which encodes the ∆12-desaturase, ∆4-desaturase, ∆5-desaturase, ∆6-desaturase, ∆5-elongase, ∆6-elongase and/or ω3-desaturase, a gene construct or a vector as described below, alone or in combination with further nucleic acid sequences which encode proteins of the fatty acid or liµl d metabolism. In a further preferred embodiment, this process furthermore comprises the step of obtaining the oils, liµl ds or free fatty acids from the seed of the plant, such as, for example, the seed of an oil crop, such as, for example, peanut, oilseed rap, canola, linseed, hemp, peanut, soybean, safflower, hemp, sunflowers or borage.
In the case of plant cells, plant tissue or plant organs, "growing" is understood as meaning, for example, the cultivation on or in a nutrient medium, or of the intact plant on or in a substrate, for example in a hydroponic culture, potting compost or on arable land.
The invention furthermore relates to gene constructs which comprise the nucleic acid sequences according to the invention which encode a ∆5-desaturase, ∆6-desaturase,

∆5-elongase or ∆6-elongase, the nucleic add being linked functionally with one or more regulatory signals. In addition, the gene construct may comprise further biosynthesis genes of the fatty acid or liµl d metabolism selected from the group consisting of acyl-CoA dehydrogenase(s), acyl-ACP [= acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyl transferase(s), acyl-CoA:lysophospholiµl d acyltransferases, fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allenoxide synthases, hydroperoxide lyases or fatty acid elongase(s). Biosynthesis genes of the fatty acid or liµl d metabolism selected from the group ∆8-desaturase, ∆9-desaturase, ∆9-elongase or ω3--desaturase are advantageously additionally present.
The nucleic acid sequences used in the process which encode proteins with ∆5-desaturase, ∆6-desaturase, ∆12-desaturase, ∆5-elongase or ∆6-elongase activity are advantageously introduced into the plant alone or, preferably, in combination with an expression cassette (= nucleic acid construct) which makes possible the expression of the nucleic acids in the plant. The nucleic acid construct can comprise more than one nucleic acid sequence with an enzymatic activity, for example, of a ∆12-desaturase, ∆5-desaturase, ∆6-desaturase, ∆5-elongase and/or ∆6-elongase.
To introduce the nucleic acids into the gene constructs, the nucleic acids used in the process are advantageously amplified and ligated in the known manner. Preferably, a procedure following the protocol for Pfu DNA polymerase or a Pfu/Taq DNA polymerase mixture is followed. The primers are selected taking into consideration the sequence to be amplified. The primers should expediently be chosen in such a way that the amplificate comprises the entire codogenic sequence from the start codon to the stop codon. After the amplification, the amplificate is expediently analyzed. For example, a gel-electrophoretic separation can be carried out, which is followed by a quantitative and a qualitative analysis. Thereafter, the amplificate can be purified following a standard protol (for example Qiagen). An aliquot of the purified amplificate is then available for the subsequent cloning step.
Suitable cloning vectors are generally known to the skilled worker. These include, in particular, vectors which are capable of replication in microbial systems, that is to say mainly vectors which ensure efficient cloning in yeasts or fungi and which make possible the stable transformation of plants. Those which must be mentioned in particular are various binary and cointegrated vector systems which are suitable for the T-DNA-mediated transformation. Such vector systems are, as a rule, characterized in

that they comprise at least the vir genes required for the Agrobacterium-mediated transformation and the T-DNA-delimiting sequences (T-DNA border). These vector systems preferably also comprise further cis-regulatory regions such as promoters and terminator sequences and/or selection markers, by means of which suitably transformed organisms can be identified. While in the case of cointegrated vector systems vir genes and T-DNA sequences are arranged on the same vector, binary systems are based on at least two vectors, one of which bears vir genes, but no T-DNA, while a second one bears T-DNA, but no vir genes. Owing to this fact, the last-mentioned vectors are relatively small, easy to manipulate and capable of replication both in £ coli and in Agrobacterium. These binary vectors include vectors from the series pBIB-HYG, pPZP, pBecks, pGreen. In accordance with the invention, Bin 19, pBI101, pBinAR, pGPTV and pCAMBIA are used by preference. An overview of the binary vectors and their use is found in Hellens et al, Trends in Plant Science (2000) 5, 446-451.
In order to prepare the vectors, the vectors can first be linearized with restriction endonuclease(s) and then modified enzymatically in a suitable manner. Thereafter, the vector is purified, and an aliquot is employed for the cloning step. In the cloning step, the enzymatically cleaved and, if appropriate, purified amplificate is ligated with vector fragments which have been prepared in a similar manner, using ligase. In this context, a particular nucleic acid construct, or vector or plasmid construct, can have one or more than one codogenic gene segments. The codogenic gene segments in these constructs are preferably linked functionally with regulatory sequences. The regulatory sequences include, in particular, plant sequences such as promoters and terminator sequences. The constructs can advantageously be stably propagated in microorganisms, in particular in £ coli and Agrobacterium tumefaciens, under selection conditions and make possible a transfer of heterologous DNA into plants or microorganisms.
The nucleic acids used in the process can be introduced into plants, advantageously using cloning vectors, and thus be used in the transformation of plants such as those which are published and cited therein: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapter 6/7, p. 71-119 (1993); F.F. White, Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, Eds.: Kung and R. Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, Eds.: Kung and R. Wu, Academic Press (1993), 128-143; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225. Thus, the nucleic acids

and/or vectors used in the process can be used for the recombinant modification of a broad spectrum of plants so that the latter become better and/or more efficient PUFA producers.
A series of mechanisms by which a modification of the ∆12-desaturase, ∆5-elongase, ∆6-elongase, ∆5-desaturase and/or ∆6-desaturase protein is possible exists, so that the yield, production and/or production efficiency of the polyunsaturated fatty acids in a plant, preferably in an oilseed plant or oil crop, can be influenced directly owing to this modified protein. The number or activity of the ∆12-Desaturase, ∆6-desaturase, ∆5-eiongase, ∆6-elongase or ∆5-desaturase proteins or genes can be increased, so that greater amounts of the gene products and, ultimately, greater amounts of the compounds of the general formula I are produced. A de novo synthesis in a plant which has lacked the activity and ability to biosynthesize the compounds prior to introduction of the corresponding gene(s) is also possible. This applies analogously to the combination with further desaturases or elongases or further enzymes of the fatty acid and liµl d metabolism. The use of various divergent sequences, i.e. sequences which differ at the DNA sequence level, may also be advantageous in this context, or else the use of promoters which make possible a different gene expression in the course of time, for example as a function of the degree of maturity of a seed or an oil-storing tissue.
Owing to the introduction of a combination of ∆12-desaturase, ∆6-desaturase, ∆5-elongase, ∆6-elongase and/or ∆5-desaturase genes into the plant, alone or in combination with other genes, it is not only possible to increase biosynthesis flux towards the end product, but also to increase, or to create de novo the corresponding triacylglycerol composition. Likewise, the number or activity of other genes which are involved in the import of nutrients which are required for the biosynthesis of one or more fatty acids, oils, polar and/or neutral liµl ds, can be increased, so that the concentration of these precursors, cofactors or intermediates within the cells or within the storage compartment is increased, whereby the ability of the cells to produce PUFAs is enhanced further. By optimizing the activity or increasing the number of one or more ∆12-desaturase, ∆6-desaturase, ∆5-elongase, ∆6-elongase or ∆5-desaturase genes which are involved in the biosynthesis of these compounds, or by destroying the activity of one or more genes which are involved in the degradation of these compounds, an enhanced yield, production and/or production efficiency of fatty acid and liµl d molecules in plants is made possible.

The nucleic acid sequences used in the process are advantageously introduced into an expression cassette which makes possible the expression of the nucleic acids in plants.
In doing so, the nucleic acid sequences which encode ∆12-desaturase, ∆6-desaturase, ∆5-elongase, ∆6~elongase or ∆5-desaturase are linked functionally with one or more regulatory signals, advantageously for enhancing gene expression. These regulatory sequences are intended to make possible the specific expression of the genes and proteins. Depending on the host organism, this may mean, for example, that the gene is expressed and/or overexpressed only after induction has taken place, or else that it is expressed and/or overexpressed immediately. For example, these regulatory sequences take the form of sequences to which inductors or repressors bind, thus controlling the expression of the nucleic acid. In addition to these novel regulatory sequences, or instead of these sequences, the natural regulatory elements of these sequences may still be present before the actual structural genes and, if appropriate, may have been genetically modified in such a way that their natural regulation is eliminated and the expression of the genes is enhanced. These modified promoters can also be positioned on their own before the natural gene in the form of part-sequences (= promotor with parts of the nucleic acid sequences used in accordance with the invention) in order to enhance the activity. Moreover, the gene construct may advantageously also comprise one or more what are known as enhancer sequences in operable linkage with the promoter, which make possible an enhanced expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminator sequences, may also be inserted at the 3' end of the DNA sequences.
The ∆12-desaturase, ∆5-desaturase, ∆6-desaturase, ∆5-elongase and/or ∆6-elongase genes may be present in one or more coµl es of the expression cassette (= gene construct). Preferably, only one copy of the genes is present in each expression cassette. This gene construct, or the gene constructs, can be expressed together in the host plant. In this context, the gene construct(s) can be inserted in one or more vectors and be present in the cell in free form, or else be inserted in the genome. It is advantageous for the insertion of further genes in the host genome when the genes to be expressed are present together in one gene construct.
In this context, the regulatory sequences or factors can, as described above, preferably have a positive effect on the gene expression of the genes introduced, thus enhancing it. Thus, an enhancement of the regulatory elements, advantageously at the

transcriptional level, may take place by using strong transcription signals such as promoters and/or enhancers. In addition, however, enhanced translation is also possible, for example by improving the stability of the mRNA.
In a further embodiment of the invention, one or more gene constructs comprising one or more sequences which are defined by SEQ ID NO: 11, SEQ ID NO: 27, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201 or their derivatives and which encode polypeptides as shown in SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202 are present. The abovementioned ∆12-desaturase, ∆6-desaturase, ∆5-elongase, ∆6-elongase or ∆5-desaturase proteins advantageously lead to a desaturation or elongation of fatty acids, the substrate advantageously having one, two, three or four double bonds and advantageously 18, 20 or 22 carbon atoms in the fatty acid molecule. The same applies to their homologs, derivatives or analogs which are linked functionally with one or more regulatory signals, preferably for enhancing gene expression.
In principle, it is possible to use all natural promoters together with their regulatory sequences, such as those mentioned above, for the novel process. It is also possible and advantageous to use synthetic promoters, either in addition or alone, in particular when they mediate seed-speciftc expression, such as those described in WO 99/16890.
In order to achieve a particularly high PUFA content, especially in transgenic plants, the PUFA biosynthesis genes should advantageously be expressed in oilseeds in a seed-specific manner. To this end, seed-specific promoters can be used, or those promoters which are active in the embryo and/or in the endosperm. In principle, seed-specific promoters can be isolated both from dicotyledonous and from monocotyledanous plants. Preferred promoters are listed hereinbelow: USP (= unknown seed protein) and vicilin (Vicia faba) [Bauµlein et al., Mol. Gen Genet., 1991, 225(3)], naµl n (oilseed rape) [US 5,608,152], conlinin (linseed) [WO 02/102970], acyl carrier protein (oilseed rape) [US 5,315,001 and WO 92/18634], oleosin (Arabidopsis thaliana) [WO 98/45461 and WO 93/20216], phaseolin (Phaseolus vulgaris) [US 5,504,200], Bce4 [WO 91/13980], legumes B4 (LegB4 promoter) [Bauµlein et al., Plant J., 2,2, 1992], Lpt2 and Ipt1 (barley) [WO 95/15389 and WO95/23230], seed-specific promoters from rice, maize and wheat [WO 99/16890], Amy32b, Amy 6-6 and aleurain [US 5,677,474], Bce4 (oilseed rape) [US 5,530,149], glycinin (soybean) [EP 571 741], phosphoenol pyruvate carboxylase (soybean) [JP 06/62870], ADR12-2 (soybean)

[WO 98/08962], isocitrate lyase (oilseed rape) [US 5,689,040] or a-amylase (barley) [EP 781 849].
Plant gene expression can also be facilitated via a chemically inducible promoter (see a review in Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Bio!., 48:89-108). Chemically inducible promoters are particularly suitable when it is desired that gene expression should take place in a time-specific manner. Examples of such promoters are a salicylic-acid-inducible promoter (WO 95/19443), a tetracyclin-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404) and an ethanol-inducible promoter.
To ensure the stable integration of the biosynthesis genes into the transgenic plant over a plurality of generations, each of the nucleic acids which encode ∆12-desaturase, ∆6-desaturase, ∆5-elongase, ∆6-elongase and/or ∆5-desaturase and which are used in the process should be expressed under the control of a separate promoter, preferably a promoter which differs from the other promoters, since repeating sequence motifs can lead to instability of the T-DNA, or to recombination events. In this context, the expression cassette is advantageously constructed in such a way that a promoter is followed by a suitable cleavage site, advantageously in a polylinker, for insertion of the nucleic acid to be expressed and, if appropriate, a terminator sequence is positioned behind the polylinker. This sequence is repeated several times, preferably three, four, five, six or seven times, so that up to seven genes can be combined in one construct and introduced into the transgenic plant in order to be expressed. Advantageously, the sequence is repeated up to four times. To express the nucleic acid sequences, the latter are inserted behind the promoter via a suitable cleavage site, for example in the polylinker. Advantageously, each nucleic acid sequence has its own promoter and, if appropriate, its own terminator sequence. Such advantageous constructs are disclosed, for example, in DE 101 02 337 or DE 101 02 338. However, it is also possible to insert a plurality of nucleic acid sequences behind a shared promoter and, if appropriate, before a shared terminator sequence. Here, the insertion site, or the sequence, of the inserted nucleic acids in the expression cassette is not of critical importance, that is to say a nucleic acid sequence can be inserted at the first or last position in the cassette without its expression being substantially influenced thereby. Advantageously, different promoters such as, for example, the USP, LegB4 or DC3 promoter, and different terminator sequences can be used in the expression cassette. However, it is also possible to use only one type of promoter in the cassette, which, however, may lead to undesired recombination events.

As described above, the transcription of the genes which have been introduced should advantageously be terminated by suitable terminator sequences at the 31 end of the biosynthesis genes which have been introduced (behind the stop codon). An example of a sequence which can be used in this context is the OCS1 terminator sequence. As is the case with the promoters, different terminator sequences should be used for each gene.
As described above, the gene construct can also comprise further genes to be introduced into the plants. It is possible and advantageous to introduce into the host plants, and to express, regulatory genes such as genes for inductors, repressors or enzymes which, owing to their enzyme activity, engage in the regulation of one or more genes of a biosynthesis pathway. These genes can be of heterologous or of homologous origin.
Moreover, further biosynthesis genes of the fatty acid or liµl d metabolism can advantageously be present in the nucleic acid construct, or gene construct; however, these genes can also be present on one or more further nucleic acid constructs. A biosynthesis gene of the fatty acid or liµl d metabolism which is preferably chosen is a gene from the group consisting of acyl-CoA dehydrogenase(s), acyl-ACP [= acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyl transferase(s), acyl-CoA:lysophospholiµl d acyltransferases, fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allenoxide synthases, hydroperoxide lyases or fatty acid elongase(s) or combinations thereof.
Especially advantageous nucleic acid sequences are biosynthesis genes of the fatty acid or liµl d metabolism selected from the group of the acyl-CoA:lysophospholiµl d acyltransferase, ω3-desaturase, ∆8-desaturase, ∆4-desaturase, ∆9-desaturase, ∆5-elongase and/or ∆9-elongase.
In this context, the abovementioned nucleic acids or genes can be cloned into expression cassettes, like those mentioned above, in combination with other elongases and desaturases and used for transforming plants with the aid of Agrobacterium.
Here, the regulatory sequences or factors can, as described above, preferably have a positive effect on, and thus enhance, the gene expression of the genes which have been introduced. Thus, enhancement of the regulatory elements can advantageously

take place at the transcriptional level by using strong transcription signals such as promoters and/or enhancers. However, an enhanced translation is also possible, for example by improving the stability of the mRNA. In principle, the expression cassettes can be used directly for introduction into the plants or else be introduced into a vector.
These advantageous vectors, preferably expression vectors, comprise the nucleic acids which encode the ∆12-desaturases, ∆6-desaturases, ∆5-eiongases, ∆6-elongases or ∆5-desaturases and which are used in the process, or else a nucleic acid construct which comprises the nucleic acid used either alone or in combination with further biosynthesis genes of the fatty acid or liµl d metabolism such as the acyl-CoA:lysophospholiµl d acyltransferases, a)3-desaturases, ∆8-desaturases, ∆9-desaturases, ω3-desaturases, ∆4-desaturases, ∆5-elongases and/or ∆9-elongases.
As used in the present context, the term "vector" refers to a nucleic acid molecule which is capable of transporting another nucleic acid to which it is bound. One type of vector is a "µl asmid", a circular double-stranded DNA loop into which additional DNA segments can be ligated. A further type of vector is a viral vector, it being possible for additional DNA segments to be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they have been introduced (for example bacterial vectors with bacterial replication origin). Other vectors are advantageously integrated into the genome of a host cell when they are introduced into the host cell, and thus replicate together with the host genome. Moreover, certain vectors can govern the expression of genes with which they are in operable linkage. These vectors are referred to in the present context as "expression vectors". Usually, expression vectors which are suitable for DNA recombinatidn techniques take the form of plasmids. In the present description, "plasmid" and "vector" can be used exchangeably since the plasmid is the form of vector which is most frequently used. However, the invention is also intended to cover other forms of expression vectors, such as viral vectors, which exert similar functions. Furthermore, the term "vector" is also intended to encompass other vectors with which the skilled worker is familiar, such as phages, viruses such as SV40, CMV, TMV, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA.
The recombinant expression vectors advantageously used in the process comprise the nucleic acids or the described gene construct used in accordance with the invention in a form which is suitable for expressing the nucleic acids used in a host cell, which means that the recombinant expression vectors comprise one or more regulatory sequences, selected on the basis of the host cells used for the expression, which

regulatory sequence(s) is/are linked functionally with the nucleic acid sequence to be expressed. In a recombinant expression vector, "linked functionally" or "in operable linkage" means that the nucleotide sequence of interest is bound to the regulatory sequence(s) in such a way that the expression of the nucieotide sequence is possible and they are bound to each other in such a way that both sequences carry out the predicted function which is ascribed to the sequence (for example in an in-vitro transcription/translation system, or in a host cell if the vector is introduced into the host cell).
The term "regulatory sequence" is intended to comprise promoters, enhancers and other expression control elements (for example polyadenylation signals). These regulatory sequences are described, for example, in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990), or see: Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, Florida, Eds.: Glick and Thompson, Chapter 7, 89-108, including the references cited therein. Regulatory sequences comprise those which govern the constitutive expression of a nucleotide sequence in many types of host cell and those which govern the direct expression of the nucleotide sequence only in specific host cells under specific conditions. The skilled worker knows that the design of the expression vector can depend on factors such as the choice of host cell to be transformed, the desired expression level of the protein and the like.
In a further embodiment of the process, the ∆12-desaturases, ∆6-desaturases, ∆5-elongases, ∆6-elongases and/or ∆5-desaturases can be expressed in single-celled plant cells (such as algae), see Falciatore et al., 1999, Marine Biotechnology 1 (3):239-251 and references cited therein, and in plant cells from higher plants (for example spermatophytes such as arable crops). Examples of plant expression vectors comprise those which are described in detail in: Becker, D., Kemper, EM Schell, J., and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol. 20:1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, Eds.: Kung and R. Wu, Academic Press, 1993, p. 15-38.
A plant expression cassette preferably comprises regulatory sequences which are capable of governing the expression of genes in plant cells and which are linked functionally so that each sequence can fulfill its function, such as transcriptional termination, for example polyadenylation signals. Preferred polyadenylation signals are

those which are derived from Agrobactehum tumefaciens T-DNA, such as gene 3 of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835 et seq.), which is known as octoµl ne synthase, or functional equivalents thereof, but all other terminator sequences which are functionally active in plants are also suitable.
Since the regulation of plant gene expression is very often not limited to the transcriptional level, a plant expression cassette preferably comprises other sequences which are linked functionally, such as translation enhancers, for example the overdrive sequence, which enhances the tobacco mosaic virus 5'-untranslated leader sequence, which increases the protein/RNA ratio (Gallie et al., 1987, Nucl. Acids Research 15:8693-8711).
As described above, the gene to be expressed must be linked functionally with a suitable promoter which triggers gene expression with the correct planning or in a cell-or tissue-specific manner. Utilizable promoters are constitutive promoters (Benfey et al., EMBO J. 8 (1989) 2195-2202), such as those which are derived from plant viruses, such as 35S CaMV (Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see also US 5352605 and WO 84/02913), or constitutive plant promoters, such as the promoter of the Rubisco small subunit, which is described in US 4,962,028.
As described above, plant gene expression can also be achieved via a chemically inducible promoter (see a review in Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89-108). Chemically inducible promoters are particularly suitable when it is desired that the gene expression takes place in a time-specific manner. Examples of such promoters are a salicylic-acid-inducible promoter (WO 95/19443), a tetracyclin-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404) and an ethanol-inducible promoter.
Promoters which respond to biotic or abiotic stress conditions are also suitable, for example the pathogen-induced PRP1 gene promoter (Ward et al., Plant. Mol. Biol. 22 (1993) 361-366), the heat-inducible tomato hsp80 promoter (US 5,187,267), the chill-inducible potato alpha-amylase promoter (WO 96/12814) or the wound-inducible µl nll promoter (EP-A-0 375 091).
Especially preferred are those promoters which bring about the gene expression in tissues and organs in which the biosynthesis of fatty acids, liµl ds and oils takes place, in seed cells, such as cells of the endosperm and of the develoµl ng embryo. Suitable promoters are the oilseed rape naµl n promoter (US 5,608,152), the linseed Conlinin

promoter (WO 02/102970), the Vicia faba USP promoter (Baeuµlein et al., Mol Gen Genet, 1991, 225 (3):459-67), the Arabidopsis oleosin promoter (WO 98/45461), the Phaseolus vulgaris phaseolin promoter (US 5,504,200), the Brassica Bce4 promoter (WO 91/13980) or the legume B4 promoter (LeB4; Baeuµlein et al., 1992, Plant Journal, 2 (2):233-9), and promoters which bring about the seed-specific expression in monocotyledonous plants such as maize, barley, wheat, rye, rice and the like. Suitable noteworthy promoters are the barley Ipt2 or Ipt1 gene promoter (WO 95/15389 and WO 95/23230) or the promoters from the barley hordein gene, the rice glutelin gene, the rice oryzin gene, the rice proiamine gene, the wheat gliadine gene, the wheat glutelin gene, the maize zeine gene, the oat glutelin gene, the sorghum kasirin gene or the rye secalin gene, which are described in WO 99/16890.
Other promoters which are also particularly suitable are those which bring about the plastid-specific expression, since plastids constitute the compartment in which precursors and some end products of liµl d biosynthesis are synthesized. Suitable promoters are the viral RNA polymerase promoter, described in WO 95/16783 and WO 97/06250, and the Arabidopsis dpP promoter, described in WO 99/46394.
In particular, it may be desired to bring about the multiparallel expression of the ∆12-desaturases, ∆6-desaturases, ∆5-elongases, ∆6-elongases and/or ∆5-desaturases used in the process. Such expression cassettes can be introduced via the simultaneous transformation of a plurality of individual expression constructs or, preferably, by combining a plurality of expression cassettes on one construct. Also, a plurality of vectors can be transformed with in each case a plurality of expression cassettes and then transferred into the host cell.
Other preferred sequences for the use in operable linkage in plant gene expression cassettes are targeting sequences which are required for targeting the gene product into its corresponding cell compartment, for example into the vacuole, the nucleus, all types of plastids, such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, the mitochondria, the endoplasmic reticulum, elaioplasts, peroxisomes and other compartments of plant cells (see a review in Kermode, Crit. Rev. Plant Sci. 15, 4 (1996) 285-423 and references cited therein).
The process according to the invention employs the nucleic acid sequences with the SEQ ID NO: 11, SEQ ID NO: 27, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201 or their derivatives or homologs which encode polypeptides.which retain the enzymatic activity of the proteins encoded by nucleic acid

sequences. These sequences, individually or in combination with the nucleic acid sequences which encode the other enzymes used, are cloned into expression constructs and used for the transformation into, and expression in, plants. Owing to their construction, these expression constructs make possible an advantageous optimal synthesis of the polyunsaturated fatty acids produced in the process according to the invention.
In a preferred embodiment, the process furthermore comprises the step of obtaining a cell or an intact plant which comprises the nucleic acid sequences used in the process, where the cell and/or the plant is transformed with a nucleic acid sequence encoding a polypeptide with a ∆12-desaturase, ∆5-desaturase, ∆6-desaturase, ∆5-elongase and/or ∆6-elongase activity, a gene construct or a vector as described above, alone or in combination with further nucleic acid sequences which encode proteins of the fatty acid or liµl d metabolism. The resulting cell is advantageously a ceil of an oil-producing organism such as an oil crop, such as, for example, peanut, oilseed rape, canola, linseed, hemp, peanut, soybean, safflower, hemp, mustard, sunflowers or borage.
For the purposes of the invention, "transgenic" or "recombinant" means with regard to, for example, a nucleic acid sequence, an expression cassette (= gene construct) or a vector comprising the nucleic acid sequence according to the invention or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either
a) the nucleic acid sequence according to the invention, or
b) a genetic control sequence which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or
c) a) and b)
are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original organism or the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks

the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp. A naturally occurring expression cassette - for example the naturally occurring combination of the natural promoter of the nucleic acid sequences used in the process according to the invention with the corresponding ∆12-desaturase, ∆4-desaturase, ∆5-desaturase, ∆6-desaturase, ∆8-desaturase, ω3--desaturase, ∆9-elongase, ∆6-elongase and/or ∆5-elongase genes - becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic ("artificial") methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in US 5,565,350 or WO 00/15815.
Transgenic plants for the purposes of the invention is therefore understood as meaning that the nucleic acids used in the process are not at their natural locus in the genome of the plant, it being possible for the nucleic acids to be expressed homologously or heterologously. However, transgenic also means that, while the nucleic acids according to the invention are at their natural position in the genome of the plant, however, the sequence having been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention or the nucleic acid sequences used in the process according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place. Preferred transgenic plants are oilseed or oil fruit crops.
Plants which are suitable for use in the process according to the invention are, in principle, advantageously all plants which are capable of synthesizing fatty acids, specifically unsaturated fatty acids such as ARA, EPA and/or DHA, and which are suitable for the expression of recombinant genes. Examples are plants such as Arabidopsis, Asteraceae such as Calendula or crop plants such as soybean, peanut, castor-oil plant, sunflower, maize, cotton, flax, oilseed rape, coconut, oil pal.,, safflower (Carthamus tinctorius) or cacao bean. Plants which are naturally capable of synthesizing large amounts of oils are preferred, such as soybean, oilseed rape, Camelina, Indian mustard, coconut, oil pal.,, safflower (Carthamus tinctorius), flax, hemp, castor-oil plant, Calendula, peanut, cacao bean or sunflower or yeast such as Saccharomyces cerevisiae, with soybean, flax, oilseed rape, safflower, sunflower, Camelina, indian mustard or Calendula being especially preferred.

Further host cells which can be used for cloning the nucleic acid sequences used in the process according to the invention are detailed in; Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
Expression strains which can be used, for example those with a lower protease activity, are described in: Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128.
These include plant cells and certain tissues, organs and parts of plants in all their phenotyµl c forms such as anthers, fibers, root hairs, stalks, embryos, calli, cotelydons, petioles, harvested material, plant tissue, reproductive tissue and cell cultures which is derived from the actual transgenic plant and/or can be used for bringing about the transgenic plant.
Transgenic plants or advantageously the seeds thereof which comprise the polyunsaturated fatty acids in particular ARA, EPA and/or DHA, synthesized in the process according to the invention can advantageously be marketed directly without there being any need for the oils, liµl ds or fatty acids synthesized to be isolated. Plants for the process according to the invention are as meaning intact plants and all plant parts, plant organs or plant parts such as leaf, stem, seeds, root, tubers, anthers, fibers, root hairs, stalks, embryos, calli, cotelydons, petioles, harvested material, plant tissue, reproductive tissue and cell cultures which are derived from the actual transgenic plant and/or can be used for bringing about the transgenic plant. In this context, the seed comprises all parts of the seed such as the seed coats, eµl dermal cells, seed cells, endosperm or embryonic tissue.
In principle, the process according to the invention is also suitable for the production of polyunsaturated fatty acids, in particular ARA, EPA and/or DHA, in plant cell cultures, followed by obtaining the fatty acids from the cultures. In particular, they may take the form of suspension or callus cultures.
However, the compound produced in the process according to the invention can also be isolated from the plants, advantageously the plant seeds, in the form of their oils, fat, liµl ds and/or free fatty acids. Polyunsaturated fatty acids produced by this process, in particular ARA, EPA and/or DHA, can be harvested by harvesting the plants or plant seeds either from the culture in which they grow, or from the field.

In a further preferred embodiment, this process furthermore comprises the step of obtaining the oils, liµl ds or free fatty acids from the plant or from the crop. The crop may, for example, take the form of a greenhouse- or field-grown plant crop.
The oils, liµl ds or free fatty acids can be isolated via pressing or extraction of the plant parts, preferably the plant seeds. In this context, the oils, fats, liµl ds and/or free fatty acids can be obtained by what is known as cold-beating or cold-pressing without applying heat. To allow for greater ease of disruption of the plant parts, specifically the seeds, they are previously comminuted, steamed or roasted. The seeds which have been pretreated in this manner can subsequently be pressed or extracted with solvents such as warm hexane. The solvent is subsequently removed.
Thereafter, the resulting products which comprise the polyunsaturated fatty acids are processed further, i.e. refined. In this process, substances such as the plant mucilages and suspended matter are first removed. What is known as desliming can be effected enzymatically or, for example, chemico-physically by addition of acid such as phosphoric acid. Thereafter, the free fatty acids are removed by treatment with a base, for example sodium hydroxide solution. The resulting product is washed thoroughly with water to remove the alkali remaining in the product and then dried. To remove the µl gment remaining in the product, the products are subjected to bleaching, for example using fuller's earth or active charcoal. At the end, the product is deodorized, for example using steam.
The PUFAs or LCPUFAs produced by this process are preferably C18-, C20- or C22-fatty acid molecules, advantageously C20- or C22-fatty acid molecules, with at least two double bonds in the fatty acid molecule, preferably with three, four, five or six double bonds, especially preferably with four, five or six double bonds. These C18-, C20- or C22-fatty acid molecules can be isolated from the plant in the form of an oil, a liµl d or a free fatty acid. Examples of suitable plants are those mentioned above. Suitable organisms are transgenic plants.
One embodiment of the invention are therefore oils, liµl ds or fatty acids or fractions thereof which have been prepared by the above-described process, especially preferably oils, liµl ds or a fatty acid composition which comprise PUFAs and originate from transgenic plants.
The fatty acids obtained in the process are also suitable as starting material for the chemical synthesis of products of value. For example, they can be used together or

alone for the production of Pharmaceuticals, foodstuffs, feedstuffs or cosmetics.
As described above, these oils, liµl ds or fatty acids advantageously comprise 6 to 15% of pal.,itic acid, 1 to 6% of stearic acid, 7-85% of oieic acid, 0.5 to 8% of vaccenic acid, 0.1 to 1% of arachic acid, 7 to 25% of saturated fatty acids, 8 to 85% of monounsaturated fatty acids and 60 to 85% of polyunsaturated fatty acids, in each case based on 100% and on the total fatty acid content of the organisms. Advantageous polyunsaturated fatty acids which are present in the fatty acid esters or fatty acid mixtures are preferably at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1% of arachidonic acid, based on the total fatty acid content. Moreover, the fatty acid esters or fatty acid mixtures which have been produced by the process of the invention advantageously comprise fatty acids selected from the group of the fatty acids erucic acid (13-docosaenoic acid), sterculic acid (9,10-methyleneoctadec-9-enoic acid), malvalic acid (8,9-methyleneheptadec-8-enoic acid), chaulmoogric acid (cyclopentenedodecanoic acid), furan fatty acid (9,12-epoxyoctadeca-9,11-dienoic acid), vernolic acid (9,10-epoxyoctadec-12-enoic acid), tariric acid (6-octadecynoic acid), 6-nonadecynoic acid, santalbic acid (t11-octadecen-9-ynoic acid), 6,9-octadecenynoic acid, pyrulic acid (t10-heptadecen-8-ynoic acid), crepenyninic acid (9-octadecen-12-ynoic acid), 13,14-dihydrooropheic acid, octadecen-13-ene-9,11-diynoic acid, petroselenic acid (cis-6-octadecenoic acid), 9c,12t-octadecadienoic acid, calendulic acid (8t10t12c-octadecatrienoic acid), catalµl c acid (9t11t13c-octadecatrienoic acid), eleostearic acid (9c11t13t-octadecatrienoic acid), jacaric acid (8c10t12c-octadecatrienoic acid), punicic acid (9c11t13c-octadecatrienoic acid), parinaric acid (9c11t13t15c-octadecatetraenoic acid), µl nolenic acid (all-cis-5,9,12-octadecatrienoic acid), laballenic acid (5,6-octadecadienallenic acid), ricinoleic acid (12-hydroxyoleic acid) and/or coriolic acid (13-hydroxy-9c,11t-octadecadienoic acid). The abovementioned fatty acids are, as a rule, advantageously only found in traces in the fatty acid esters or fatty acid mixtures produced by the process according to the invention, that is to say that, based on the total fatty acids, they occur to less than 30%, preferably to less than 25%, 24%, 23%, 22% or 21%, especially preferably to less than 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5%, very especially preferably to less than 4%, 3%, 2% or 1%. In a further preferred form of the invention, these abovementioned fatty acids occur in amounts of less than 0.9%, 0.8%, 0.7%, 0.6% or 0.5%, especially preferably less than 0.4%, 0.3%, 0.2%, 0.1%, based on the total fatty acids. The fatty acid esters or fatty acid mixtures produced by the process according to the invention advantageously comprise less than 0.1%, based on the total fatty acids, and/or no butyric acid, no cholesterol, no clupanodonic acid (= docosapentaenoic acid, C22.5∆4,8,i2,i5,2i) and no njsjnjc acjd (tetracosahexaenoic acid, C23:6A3-8'12'1511821).

As a rule, the abovementioned fatty acids are advantageously only found in traces in the fatty acid esters or fatty acid mixtures produced by the process according to the invention, that is to say that, based on the total fatty acids, they are found in amounts of less than 30%, preferably less than 25%, 24%, 23%, 22% or 21%, especially preferably less than 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5%, very especially preferably less than 4%, 3%, 2% or 1%. In a further preferred embodiment of the invention, these abovementioned fatty acids are found relative to the total fatty acids in amounts of less than 0.9%, 0.8%, 0.7%, 0.6% or 0.5%, especially preferably less than 0.4%, 0.3%, 0.2%, 0.1 %. The fatty acid esters or fatty acid mixtures produced by the process according to the invention advantageously comprise less than 0.1% based on the total fatty acids and/or no butyric acid, no cholesterol, no clupanodonic acid (= docosapentaenoic acid, c22:5∆4i8i12f15'21) and no nisinic acid (tetracosahexaenoic acid,
P23'6∆3'8.122h
The oils, liµl ds or fatty acids according to the invention advantageously comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, advantageously at least 11%, 12%, 13%, 14%, 15%, 16% or 17%, especially advantageously at least 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% of ARA or at least 0.5%, 1%, 2%, 3%, 4%, 5% or 6%, advantageously at least 7%, 8%, 9%, 10% or 11%, especially advantageously at least 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of EPA or at least 0.01 %, 0.02%, 0.03%, 0.04% or 0.05% or 0.06%, advantageously at least 0.07%, 0.08%, 0.09% or 0.1%, especially advantageously at least 0.2%, 0.3% or 0.4% of DHA, based on the total fatty acid content of the production organism, advantageously of a plant, especially advantageously of an oil crop such as soybean, oilseed rape, coconut, oil pal.,, safflower, flax, hemp, castor-oil plant, Calendula, peanut, cacao bean, sunflower or the abovementioned other monocotyledonous or dicotyledonous oil crops. All percentages are by weight.
Owing to the nucleic acid sequences according to the invention, or the nucleic acid sequences used in the process according to the invention, it is possible to obtain an increase in the yield of polyunsaturated fatty acids, mainly ARA and EPA, but also DHA, of at least 50, 80 or 100%, advantageously at least 150, 200 or 250%, especially advantageously at least 300, 400, 500, 600, 700, 800 or 900%, very advantageously at least 1000, 1100, 1200, 1300, 1400 or 1500% in comparison with the non-transgenic starting plant, for example a plant such as Brassica juncea, Brassica napus, Camelina sativa, Arabidopsis thanliana or Linum usitatissimum when using a GC analysis for comparison purposes, see Examples.

The liµl cls and/or oils produced in the process according to the invention have a higher content of the unsaturated fatty acids oleic acid, iinoleic acid and a-linolenic acid in the sn2-position in comparison with the other positions sn1 and sn3. A higher content is understood as meaning ratios of (sn1:sn2:sn3) 1:1.1:1, 1:1.5:1 to 1:3:1. Also, the arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid produced in the process likewise show, in the liµl ds and/or oils, a preference for the sn2-position in the triglyceride in comparison with the positions sn1 and sn3 of advantageously 1:1.1:1, 1:1.5:1 to 1:3:1.
As described above, the polyunsaturated C20- and/or C22-fatty acids, produced in the process, with four, five or six double bonds in the molecule will in the seed of plants which comprise no, or only very small amounts, of C12:0- or C14:0-fatty acids. Even shorter saturated fatty acids such as the fatty acids C4:0, C6:0, C8:0 or C10:0, too, should not be present in the liµl d and/or oil, or only in small amounts. Only small amounts are understood as meaning, advantageously, amounts which, when analyzed by GC, advantageously amount to less than 5, 4, 3, 2 or 1%, advantageously less than 0.9, 0.8, 0.7, 0.6 or 0.5%, especially advantageously less than 0.4, 0.3, 0.2 or 0.1%, very especially preferably less than 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.01 units GC peak area. The fatty acid C16:0 should advantageously be in the range of from 1 to 28% units GC peak area. Advantageously, the fatty acid C16:0 should be present in amounts of less than 25%, 20%, 15% or 10%, advantageously less than 9%, 8%, 7%, 6% or 5%, especially advantageously of less than 4%, 3%, 2% or 1% units GC peak area or not at all in the iiµl ds, oils and/or free fatty acids. The fatty acid C16:1 should advantageously amount to less than 1, 0.5, 0.4, 0.3, 0.2 or 0.1%, especially advantageously 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.01 units GC peak area. Very especially preferably, the fatty acid C16:1 should not be present in the oils and/or liµl ds produced in the process. The same applies to the fatty acids C15:0, C17:0, C16:1∆3trans, C16:4∆4'71013 and C18:5∆3'6'9'12'15. Besides oleic acid (C18:1∆9), the isomers (C18:1∆7, C18:1∆11) may also be present in the liµl ds, oils or free fatty acids. Advantageously in amounts of less than 5%, 4%, 3%, 2% or 1%, measured as units GC peak area. Each of the fatty acids C20:0, C20:1, C24:0 and C24:1 should be present in a range of from 0 to 1%, 0 to 3% and 0 to 5% units GC peak area, respectively. Moreover, little dihomo-y-linolenic acid (= DGLA) in terms of units GC peak area should be detectable in the seed oil and/or seed liµl d in the GC analysis. Little is understood as meaning less than 2, 1.9, 1.8, 1.7, 1.6 and 1.5%, advantageously less than 1.4, 1.3, 1.2, 1.1 or 1%, especially advantageously less than 0.9, 0.8, 0.7, 0.6, 0.5 or 0.4% in terms of units GC peak area.

In a preferred embodiment of the process, DGLA and ARA should be produced in a ratio of from 1:1 up to 1:100, advantageously 1:2 up to 1:80, especially advantageously 1:3 up to 1:70r very especially preferably 1:5 up to 1:60.
In a further preferred embodiment of the process, DGLA and EPA should be produced in a ratio of from 1:1 up to 1:100, advantageously 1:2 up to 1:80, especially advantageously 1:3 up to 1:70, very especially preferably 1:5 up to 1:60.
The liµl ds, oils and/or free fatty acids produced in the process according to the invention should advantageously have a high content of unsaturated fatty acids, advantageously of polyunsaturated acids, of at least 30, 40 or 50% by weight, advantageously of at least 60, 70 or 80% by weight, based on the total fatty acid content in the seeds of the transgenic plants.
All saturated fatty acids together should advantageously only account for a small amount in the liµl ds, oils and/or free fatty acids, preferably used plants. In this context, a small amount is understood as meaning an amount of less than 15%, 14%, 13%, 12%t 11% or 10%, preferably less than 9%, 8%, 7% or 6% in units GC peak area.
Liµl ds, oils and/or free fatty acids produced in the process should advantageously have an erucic acid content of less than 2% by weight based on the total fatty acid content of the plant. Advantageously, no erucic acid should be present in the liµl ds and/or oils. Also, the content of saturated fatty acids C16:0 and/or C18:0 should advantageously be less than 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10% by weight, advantageously less than 9, 8,7, 6 or 5% by weight, based on the total fatty acid content of the iiµl ds and/or oils. Also, longer fatty acids such as C20:0 or C22:1 should not be present at all or only in small amounts of advantageously less than 4, 3, 2 or 1% by weight, advantageously less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1% by weight, based on the total fatty acid content of the liµl ds and/or oils. Tyµl cally, no, or only small amounts, of C16:1 are present as fatty acid in the liµl ds and/or oils produced in the process according to the invention. Small amounts are advantageously understood as meaning fatty acid contents of less than 4, 3, 2 or 1% by weight, advantageously less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1% by weight, based on the total fatty acid content of the liµl ds and/or oils.
The oils, liµl ds, fatty acids or fatty acid mixtures according to the invention which are obtained after pressing are referred to as what is known as crude oils. They still

comprise all of the oil and/or liµl d contents and also compounds which are soluble in these. Such compounds are the various tocopherols such as a-tocopherol, p-tocopherol, γ-tocopherol and/or 5-tocopherol or phytosterols such as brassicasterol, campesterol, stigmasterol, (3-sitosteroI, sitostanol, ∆5-avenasterol, ∆5,24-stigmastadienol, ∆7-stigmastemol or ∆7-avenasterol. These compounds are present in a range of from 1 to 1000 mg/100 g, advantageously 10 to 800 mg/100 g of liµl d or oil. Triterpenes such as germaniol, amyrin, cycloartenol and others may also be present in these liµl ds and oils. These liµl ds and/or oils comprise the polyunsaturated fatty acids produced in the process, such as ARA, EPA and/or DHA, bound in polar and unpolar liµl ds such as phospholiµl ds, for example phosphatidylcholine, phosphatidylethanolamine, phosphatidiylinositol, phosphatidylserine, phosphatidylglycerol, galactofiµl ds, monoglycerides, diglycerides or triglycerides, to mention but a few. Lysophospholiµl ds may also be present in the liµl ds and/or oils. These components of the liµl ds and/or oils can be separated from one another by suitable processes. Cholesterol is not present in these crude oils.
A further embodiment according to the invention is the use of the oil, liµl d, fatty acids and/or the fatty acid composition in feedstuffs, foodstuffs, cosmetics or Pharmaceuticals. The oils, liµl ds, fatty acids or fatty acid mixtures according to the invention can be used in the manner with which the skilled worker is familiar for mixing with other oils, liµl ds, fatty acids or fatty acid mixtures of animal origin such as, for example, fish oils. Tyµl cal of such fish oils short-chain fatty acids such as C12:0, C14;0, C14:1, branched C15:0, C15:0, C16:0 or C16:1. Polyunsaturated C16-fatty acids such asC16:2, C16:3 orC16:4, branched C17:0, C17:1, branched C18:0 and C19:0 and also C19:0 and C19:1 are also found in fish oil. Such fatty acids are tyµl cal offish oils and are only found rarely, or not at all, in vegetable oils. Economically relevant fish oils are, for example, anchovy oil, menhaden oil, tuna oil, sardine oil, herring oil, mackerel oil, whale oil and sal.,on oil. These liµl ds and/or oils of animal origin can be used for mixing with the oils according to the invention in the form of crude oils, i.e. in the form of liµl ds and/or oils which have not yet been purified, or else various purified fractions may be used for mixing.
A further embodiment according to the invention is the use of the oil, liµl d, fatty acids and/or fatty acid compositions in feedstuffs, foodstuffs, cosmetics or Pharmaceuticals.
The oils, liµl ds, fatty acids or fatty acid mixtures according to the invention can be used in the manner with which the skilled worker is familiar for mixing with other oils, iiµl ds, fatty acids or fatty acid mixtures of animal origin such as, for example, fish oils. Again,

these oils, liµl ds, fatty acids or fatty acid mixtures, which are composed of vegetable and animal constituents, may be used for the preparation of foodstuffs, feedstuffs, cosmetics or Pharmaceuticals.
The term "oil", "liµl d" or "fat" is understood as meaning a fatty acid mixture comprising unsaturated or saturated, preferably esterified, fatty acid(s). The oil, liµl d or fat is preferably high in polyunsaturated free or, advantageously, esterified fatty acid(s), in particular linoleic acid, y-linolenic acid, dihomo-y-linolenic acid, arachidonic acid, a-linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid. The amount of unsaturated esterified fatty acids preferably amounts to approximately 30%, a content of 50% is more preferred, a content of 60%, 70%, 80%, 85% or more is even more preferred. For the analysis, the fatty acid content can, for example, be determined by gas chromatography after converting the fatty acids into the methyl esters by transesterification. The oil, liµl d or fat can comprise various other saturated or unsaturated fatty acids, for example calendulic acid, pal.,itic acid, pal.,itoleic acid, stearic acid, oleic acid and the like. The content of the various fatty acids in the oil or fat can vary, in particular depending on the starting organism.
The polyunsaturated fatty acids with advantageously at least two double bonds which are produced in the process are, as described above, for example sphingoliµl ds, phosphoglycerides, liµl ds, glycoliµl ds, phospholiµl ds, monoacylglycerol, diacylglycerol, triacylglycerol or other fatty acid esters.
Starting from the polyunsaturated fatty acids with advantageously at least five or six double bonds, which acids have been prepared in the process according to the invention, the polyunsaturated fatty acids which are present can be liberated for example via treatment with alkali, for example aqueous KOH or NaOH, or acid hydrolysis, advantageously in the presence of an alcohol such as methanol or ethanol, or via enzymatic cleavage, and isolated via, for example, phase separation and subsequent acidification via, for example, H2SO4. The fatty acids can also be liberated directly without the above-described processing step.
Mosses and algae are the only known plant systems which produce substantial amounts of polyunsaturated fatty acids such as arachidonic acid (ARA) and/or eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA). Mosses comprise PUFAs in membrane liµl ds, while algae, organisms which are related to algae and a few fungi also accumulate substantial amounts of PUFAs in the triacylglycerol fraction.

This is why nucleic acid molecules which are isolated from such strains which also accumulate PUFAs in the triacylglycerol fraction are particularly advantageous for the process according to the invention and thus for the modification of the liµl d and PUFA production system in a host, in particular plants such as oil crops, for example oilseed rape, canola, linseed, hemp, soybeans, sunflowers and borage. They can therefore be used advantageously in the process according to the invention. After their introduction into a plant cell or plant, the nucleic acids used in the process can either be present on a separate plasmid or, advantageously, integrated into the genome of the host cell. In the case of integration into the genome, integration can be random or else be effected by recombination such that the native gene is replaced by the copy introduced, whereby the production of the desired compound by the cell is modulated, or by the use of a gene in trans, so that the gene is linked operably with a functional expression unit which comprises at least one sequence which ensures the expression of a gene and at least one sequence which ensures the polyadenylation of a functionally transcribed gene. The nucleic acids are advantageously introduced into the organisms via multiexpression cassettes or constructs for multiparallel expression, advantageously into the plants for the multiparallel seed-specific expression of genes.
Naturally, the coexpression of a plurality of genes can be effected not only by introducing the genes on a shared recombinant nucleic acid construct. Rather, individual genes can also be introduced separately - simultaneously or in succession, on a variety of constructs. In this case, the simultaneous presence in the plant which coexpresses all of the genes is ensured by using different selection markers. This plant can be the product of one or more transformation procedures, or else be a hybridization product of plants comprising one or more of the genes.
Substrates which are advantageously suitable for the nucleic acids which are used in the process according to the invention and which encode polypeptides with ω3-desaturase, ∆4-desaturase, ∆5-desaturase, ∆6-desaturase, ∆8-desaturase, ∆12-desaturase, ∆5-elongase, ∆6-elongase and/or ∆9-elongase activity and/or the further nucleic acids used, such as the nucleic acids which encode polypeptides of the fatty acid or liµl d metabolism selected from the group acyl-CoA dehydrogenase(s), acyl-ACP [= acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyltransferase(s), acyl-CoA:lysophospholiµl d acyltransferase(s), fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allenoxide synthases, hydroperoxide lyases or fatty acid elongase(s) are advantageously C16-, C18-, C20- or C22-fatty acids. The fatty

acids converted as substrates in the process are preferably converted in the form of their acyl-CoA esters and/or their phospholiµl d esters. It is advantageous to use, in the process, desaturases with specificity for the acyl-CoA esters. The advantage here is that a substitution between the phospholiµl d esters, which are generally the substrate of the desaturation, and the acyl-CoA esters, can be dispensed with. Thus, a further enzyme step which, as has been shown, is limiting in some cases, can be dispensed with.
To produce the long-chain PUFAs according to the invention, the polyunsaturated Ci6-or C18-fatty acids must first be desaturated by the enzymatic activity of a desaturase and subsequently be elongated by at least two carbon atoms via an elongase. After one elongation cycle, this enzyme activity gives C18- or C20-fatty acids and after two elongation cycles C20- or C22-fatty acids. The activity of the desaturases and elongases used in the process according to the invention preferably leads to C1£r, C20- and/or C22-fatty acids, advantageously with at least two double bonds in the fatty acid molecule, preferably with three, four, five or six double bonds, especially preferably to give C20- and/or C22-fatty acids with at least three double bonds in the fatty acid molecule, preferably with three, four, five or six double bonds, very specially preferably with four, five or six double bonds in the molecule. Products of the process according to the invention which are especially preferred are arachidonic acid, eicosapentaenoic acid and/or docosahexaenoic acid. The C18-fatty acids with at least two double bonds in the fatty acid can be elongated by the enzymatic activity according to the invention in the form of the free fatty acid or in the form of the esters, such as phospholiµl ds, glycoliµl ds, sphingoliµl ds, phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol.
The preferred biosynthesis site of the fatty acids, oils, liµl ds or fats in the plants which are advantageously used is, for example, in general the seed or cell strata of the seed, so that seed-specific expression of the nucleic acids used in the process makes sense. However, it is obvious that the biosynthesis of fatty acids, oils or liµl ds need not be limited to the seed tissue, but can also take place in a tissue-specific manner in all the other parts of the plant, for example in eµl dermal cells or in the tubers.
Owing to the use of the nucleic acids according to the invention which encode a ∆5-elongase, the polyunsaturated fatty acids produced in the process can be increased by at least 5%, preferably by at least 10%, especially preferably by at least 20%, very especially preferably by at least 50% in comparison with the wild type of the organisms which do not comprise the nucleic acids recombinantly.

In principle, the polyunsaturated fatty acids produced by the process according to the invention in the plants used in the process can be increased in two different ways. Either the pool of free polyunsaturated fatty acids and/or the content of the esterified polyunsaturated fatty acids produced via the process can be enlarged. Advantageously, the pool of esterified polyunsaturated fatty acids in the transgenic organisms is enlarged by the process according to the invention.
A further subject matter according to the invention are isolated nucleic acid sequences which encode polypeptides with ∆5-elongase, the ∆5-elongases encoded by the nucleic acid sequences converting C20-fatty acids having at least four double bonds in the fatty acid molecule; which are advantageously ultimately incorporated into diacylglycerides and/or triacylglycerides.
A further subject matter of the invention is thus an isolated nucleic acid sequence which encodes polypeptides with ∆5-elongase and which has the sequence shown in SEQIDNO: 197.
A further subject matter of the invention is an isolated nucleic acid sequence which encodes polypeptides with ∆6~elongase activity and which has the sequence shown in SEQIDNO: 199.
Yet a further subject matter of the invention is an isolated nucleic acid sequence which encodes polypeptides with ∆6-desaturase activity and which has the sequence shown in SEQIDNO: 201.
The subject matters of the invention likewise extend to a recombinant nucleic acid molecule comprising:
a) one or more coµl es of a promoter which is active in plant cells, preferably in seed cells,
b) at least one nucleic acid sequence with the sequence shown in SEQ ID NO: 193 or SEQ ID NO: 201 which encodes a ∆6-desaturase activity,
c) at least one nucleic acid sequence with the sequence shown in SEQ ID NO: 11 which encodes a ∆5-desaturase activity,
d) at least one nucleic acid sequence with the sequence shown in SEQ ID NO: 27 or SEQ ID NO: 199 which encodes a ∆6-elongase activity, and
e) one or more coµl es of a terminator sequence.

Advantageously, an additional nucleic acid sequence with the sequence shown in SEQ ID NO: 195 and which encodes a ∆12-desaturase may also advantageously be present in the recombinant abovementioned nucleic acid molecule.
In a further advantageous embodiment, an additional nucleic acid sequence with the sequence shown in SEQ ID NO: 197 and which encodes a ∆5-elongase may also be present in the recombinant nucleic acid molecule.
Besides these abovementioned sequences, further biosynthetic genes of the fatty acid or liµl d metabolism selected from the group consisting of acyl-CoA dehydrogenase(s), acyl-ACP [= acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyltransferase(s), acyl-CoA:lysophospholiµl d acyltransferase(s), fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allenoxide synthases, hydroperoxide lyases or fatty acid elongase(s) may also be introduced into the recombinant nucleic acid molecule.
These genes are by preference genes of the fatty acid or liµl d metabolism selected from the group consisting of ∆4-desaturase, ∆8-desaturase, ∆9-desaturase or ∆9-elongase.
Yet a further subject matter of the invention are gene constructs which comprise the nucleic acid sequences SEQ ID NO: 11, SEQ ID NO: 27, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199 or SEQ ID NO: 201 according to the invention, the nucleic acid being functionally linked to one or more regulatory signals.
All of the nucleic acid sequences used in the process according to the invention are advantageously derived from a eukaryotic organism such as a plant, a microorganism such as an alga or an animal. By preference, the nucleic acid sequences are derived from the order Sal.,oniformes, Xenopus or Ciona, algae such as Mantoniella, Crypthecodinium, Euglena or Ostreococcus, fungi such as the genus Phytophtora or from diatoms such as the genera Thalassiosira or Phaeodactylum.
The nucleic acid sequences used in the process which encode proteins with ω3-~ desaturase, ∆4-desaturase, ∆5-desaturase, ∆6-desaturase, ∆8-desaturase, ∆9-desaturase, ∆12-desaturase, ∆5-elongase, ∆6-elongase or ∆9-elongase activity are

advantageously introduced by themselves or by preference in combination with an expression cassette (= nucleic acid construct) which the expression of the nucleic acids in a plant. More than one nucleic acid sequence of an enzymatic activity such as, for example, a ∆12-desaturase, ∆4-desaturase, ∆5-desaturase, ∆6-desaturase, ∆5-elongase, ∆6-elongase and/or ω3-desaturase may be present in the nucleic acid construct.
For introduction into the plant, the nucleic acids used in the process are advantageously subjected to amplification and ligation in the known manner as described above.
A series of mechanisms exist which enable a modification of the ∆12-desaturase, ∆5-elongase, ∆6-elongase, ∆5-desaturase, ∆4-desaturase, ∆6-desaturase and/or ω3--desaturase protein according to the invention and of the further proteins used in the process, such as the ∆12-desaturase, ∆9-elongase, ∆6-desaturase, ∆8-desaturase, ∆6-elongase, ∆5-desaturase or ∆4-desaturase proteins, so that the yield, production and/or production efficiency of the advantageously polyunsaturated fatty acids in a plant, preferably in an oil crop plant, can be influenced directly as the result of this modified protein. The number or activity of the ∆12-desaturase, ω3--desaturase, ∆9-elongase, ∆6-desaturase, ∆8-desaturase, ∆6-elongase, ∆5-desaturase, ∆5-elongase or ∆4-desaturase proteins or genes can be increased so that larger amounts of the gene products and thus ultimately larger amounts of the compounds of the general formula I are produced. A de-novo synthesis in a plant which had lacked the activity and ability to biosynthesize the compounds prior to the introduction of the gene(s) in question is also possible. The same also applies analogously to the combination with further desaturases or elongases or further enzymes from the fatty acid and liµl d metabolism. Also, the use of different, divergent sequences, i.e. sequences which differ at the DNA sequence level, may be advantageous, or the use of promoters for gene expression which makes possible a different temporal gene expression, for example depending on the degree of maturity of a seed or oil-storing tissue.
By introducing a ∆12-desaturase, ω3-desaturase, ∆9-elongase, ∆6-desaturase, ∆8-desaturase, ∆6-elongase, ∆5-desaturase, ∆5-elongase and/or ∆4-desaturase gene into a plant alone or in combination with other genes into a cell may not only increase the biosynthetic flux towards the end product, but also increase the corresponding triacylglycerol composition or create it de novo. Likewise, the number or activity of other genes in the import of nutrients required for the biosynthesis of one or more fatty acids, oils, polar and/or neutral liµl ds may be increased, so that the concentration of

NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 183, SEQ ID NO: 193, SEQ ID NO: 197, SEQ ID NO: 199 or SEQ ID NO: 201.
The nucleic acid sequences used in the process are advantageously introduced in an expression cassette which enables the expression of the nucleic acids in organisms such as microorganisms or plants.
In this context, the nucleic acid sequences which encode the ∆12-desaturase, ω3-desaturase, ∆9-elongase, ∆6-desaturase, ∆8-desaturase, ∆6-elongase, ∆5-desaturase, ∆5-elongase or ∆4-desaturase are advantageously linked functionally with one or more regulatory signals to increase gene expression. These regulatory sequences should enable the targeted expression of the genes and protein expression. For example, this may mean, depending on the host plant, that the gene is expressed and/or overexpressed only after induction has taken place, or else that it is expressed and/or overexpressed immediately. For example, these regulatory sequences take the form of sequences to which inductors or repressors bind and thus regulate the expression of the nucleic acid. In addition to these new regulatory sequences, or instead of these sequences, the natural regulation of these sequences may still be present before the actual structural genes and, if appropriate, may have been genetically modified in such a way that the natural regulation has been switched off and the expression of the genes enhanced. The expression cassette (= expression construct = gene construct) may, however, also be simpler in construction, that is to say no additional regulatory signals were inserted before the nucleic acid sequence or its derivatives, and the natural promoter together with its regulation was not removed. Instead, the natural regulatory sequence was mutated in such a way that regulation no longer takes place and/or gene expression is enhanced. These modified promoters can be placed before the natural gene in order to increase the activity either in the form of part-sequences (= promoter with parts of the nucleic acid sequences according to the invention) or else alone. Moreover, the gene construct can advantageously also comprise one or more what are known as "enhancer sequences" in functional linkage

with the promoter, and these enable an increased expression of the nucleic acid sequence. Also, it is possible to insert additional advantageous sequences at the 31 end of the DNA sequences, such as further regulatory elements or terminators. The ∆12-desaturase, ω3--desaturase, ∆4-desaturase, ∆5-desaturase, ∆6-desaturase, ∆8-desaturase, ∆5-elongase, ∆6-elongase and/or ∆9-elongase genes can be present in the expression cassette (= gene construct) as one or more coµl es. Advantageously, only in each case one copy of the genes is present in the expression cassette. This gene construct, or the gene constructs, can be expressed together in the host organism. In this context, the gene construct(s) can be inserted in one or more vectors and be present in the cell in free form or else inserted in the genome. It is advantageous for the insertion of further genes in the host genome when the genes to be expressed are present together in one gene construct.
In this context, the regulatory sequences or factors can, as described above, preferably have a positive effect on the gene expression of the genes which have been introduced, thus increasing it. Thus, enhancement of the regulatory elements can advantageously take place at the transcription level by using strong transcription signals such as promoters and/or enhancers. Besides, however, an enhancement of the translation is also possible, for example by improving the stability of the mRNA.
Advantageous regulatory sequences for the new process are present for example in promoters such as the plant promoters CaMV/35S [Franck et al., Cell 21 (1980) 285-294], PRP 1 [Ward et al., Plant Mol. Biol. 22 (1993)], SSU, OCS, Iib4, usp, STLS1, B33, nos or in the ubiquitin or phaseolin promoter. Also advantageous in this context are inducible promoters, such as the promoters described in EP-A-0 388 186 (benzylsulfonamide-inducible), Plant J. 2, 1992:397-404 (Gatz et al., tetracyclin-inducible), EP-A-0 335 528 (abscisic-acid-inducible) or WO 93/21334 (ethanol- or cyclohexenol-inducible). Further suitable plant promoters are the promoter of cytosolic FBPase or the ST-LSI promoter from potato (Stockhaus etal., EMBO J. 8, 1989, 2445), the phosphoribosyl-pyrophosphate amidotransferase promoter from Glycine max (Genbank accession No. U87999) or the node-specific promoter described in EP-A-0 249 676. Especially advantageous promoters are promoters which enable the expression in tissues which are involved in the biosynthesis of fatty acids. Very especially advantageous are seed-specific promoters such as the USP promoter in accordance with the practice, but also other promoters such as the LeB4, DC3, phaseolin or naµl n promoters. Further especially advantageous promoters are seed-specific promoters which can be used for monocotyledonous or dicotyledonous plants and which are described in US 5,608,152 (naµl n promoter from oilseed rape), WO

98/45461 (oleosin promoter from Arobidopsis), US 5,504,200 (phaseolin promoter from Phaseolus vulgaris), WO 91/13980 (Bce4 promoter from Brassica), by Baeuµlein et al., Plant JM 2, 2, 1992:233-239 (LeB4 promoter from a legume), these promoters being suitable for dicots. The following promoters are suitable for example for monocots: lpt-2 or IpM promoter from barley (WO 95/15389) and WO 95/23230), hordein promoter from barley and other promoters which are suitable and which are described in WO 99/16890.
In principle, it is possible to use all natural promoters together with their regulatory sequences, such as those mentioned above, for the novel process. Likewise, it is possible and advantageous to use synthetic promoters, either additionally or alone, especially when they mediate a seed-specific expression, such as, for example, as described in WO 99/16890.
To obtain a particularly high PUFA content especially in transgenic plants, the PUFA biosynthesis genes should advantageously be expressed in a seed-specific manner in oilseed crops. To this end, it is possible to use seed-specific promoters or those promoters which are active in the embryo and/or in the endosperm. In principle, seed-specific promoters can be isolated both from dicotyledonous and from monocotyledonous plants. Such advantageous promoters are detailed further above, for example the USP, Viciiin, Naµl n, Oleosin, Phaseolin, Bce4, LegB4, Lpt2, Ipt1, Amy32b, Amy 6-6, Aleurain or Bce4 promoter.
Moreover, chemically inducible promoters are also advantageously useful in the process according to the invention.
Further advantageous promoters which are advantageously suitable for expression in soybean are the promoters of the β-conglycinin a-subunit, of the β-conglycinin β-subunit, of the Kunitz trypsin inhibitor, of annexin, of glysinin, of albumin 2S, of legumin A1, of legumin A2 and that of BD30.
Especially advantageous promoters are the USP, LegB4, Fad3, SBP, DC-3 or cruciferin820 promoter.
Advantageous regulatory sequences which are used for the expression of the nucleic acid sequences used in the process according to the invention are terminators for the expression advantageously in soybean are Leg2A3\ Kti3\ Phas3', BD30 31 or AIS3'.

Especially advantageous terminators are the ∆7T, OCS, LeB3T or cat terminator.
To ensure a stable integration of the biosynthetic genes in the transgenic plant over several generations, each of the nucleic acids used in the process and which encodes ∆12-desaturase, ω3-desaturase, ∆9-e)ongase, ∆6-desaturase, ∆8-desaturase, ∆6-elongase, ∆5-desaturase, ∆5-elongase and/or ∆4-desaturase should, as described above, be under the control of its own promoter, preferably of a different promoter, since repeating sequence motifs can lead to instability of the T-DNA, or to recombination events. As described above, the gene construct can also comprise further genes which are to be introduced into the plant.
In this context, the regulatory sequences or factors used advantageously for the expression of the nucleic acids used in the process according to the invention can, as described above, preferably have a positive effect on the gene expression of the genes introduced.
These advantageous vectors, preferably expression vectors, comprise the nucleic acids used in the process which encode the ∆12-desaturases, ω3-desaturases, ∆9-elongases, ∆6-desaturases, ∆8-desaturases, ∆6-elongases, ∆5-desaturases, ∆5-elongases or ∆4-desaturases, or a nucleic acid construct which the used nucleic acid alone or in combination with further biosynthesis genes of the fatty acid or liµl d metabolism such as the acyl-CoA:lysophospholiµl d acyltransferases, ω3--desaturases, ∆4-desaturases, ∆5-desaturases, ∆6-desaturases, ∆8-desaturases, ∆9-desaturases, ∆12-desaturases, ω3--desaturases, ∆5-elongases, ∆6-elongases and/or ∆9-elongases.
As described and used in the present context, the term "vector" refers to a nucleic acid molecule which is capable of transporting another nucleic acid to which it is bound.
The recombinant expression vectors used can be designed for expressing ∆12-desaturases, io3-desaturases, ∆9-elongases, ∆6-desaturases, ∆8-desaturases, ∆6-elongases, ∆5-desaturases, ∆5-elongases and/or ∆4-desaturases in prokaryotic or eukaryotic cells. This is advantageous since, for the sake of simplicity, intermediate steps of the vector construction are frequently carried out in microorganisms. For example, the ∆12-desaturase, ω3--desaturase, ∆9-elongase, ∆6-desaturase, ∆8-desaturase, ∆6-elongase, ∆5-desaturase, ∆5-elongase and/or ∆4-desaturase genes can be expressed in bacterial cells, insect cells (using baculovirus expression vectors), yeast cells and other fungal cells (see Romanos, MA, et al. (1992) "Foreign gene expression in yeast: a review", Yeast 8:423-488; van den Hondel, C.A.M J.J., et

gene expression from the pET 11 d vector is based on the transcription of a T7-gn10-lac fusion promoter, which is mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is provided by the host strains BL21 (DE3) or HMS174 (DE3) from a resident ^-prophage which harbors a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.
The skilled worker is familiar with other vectors which are suitable in prokaryotic organisms, these vectors are, for example E. coli, pLG338, pACYC184, the pBR series such as pBR322, the pUC series such as pUC18 or pUC19, the M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, plN-111113-B1, X,gt11 or pBdCI, in Streptomyces µl JtOI, plJ364, plJ702 orplJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pS∆77 or pAJ667.
In a further embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression in the yeast S. cerevisiae comprise pYeDesaturased (Baldari et al. (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123) and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and processes for the construction of vectors which are suitable for use in other fungi, such as the filamentous fungi, comprise those which are described in detail in: van den Hondel, C.A.M.J.J., & Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, J.F. Peberdy et al., Ed. pp. 1-28, Cambridge University Press: Cambridge, or in: More Gene Manipulations in Fungi [J.W. Bennett & L.L. Lasure, Ed.,pp. 396-428: Academic Press: San Diego]. Further suitable yeast vectors are, for example, pAG-1, YEp6, YEp13 or pEMBLYe23.
As an alternative, the ∆12-desaturases, (jd3-desaturases, ∆9-elongases, ∆6-desaturases, ∆8-desaturases, ∆6-elongases, ∆5-desaturases, ∆5-elongases and/or ∆4-desaturases can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors which are available for the expression of proteins in cultured insect cells (for example Sf9 cells) comprise the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
The abovementioned vectors are only a small overview of possible suitable vectors. Further plasmids are known to the skilled worker and are described, for example, in: Cloning Vectors (Ed., Pouwels, P.H., et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). Further suitable expression systems for prokaryotic and eukaryotic cells, see the chapters 16 and 17 of Sambrook, J., Fritsch, E.F., and

Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
To detect the enzyme activity, ∆12-desaturases, io3-desaturases, ∆9-elongases, ∆6-desaturases, ∆8-desaturases, ∆6-elongases, ∆5-desaturases, ∆5-elongases and/or ∆4-desaturases can be expressed in single-cell plant cells (such as algae), see Falciatore et al., 1999, Marine Biotechnology 1 (3):239-251 and the references cited therein, and plant cells from higher plants (for example Spermatophytes, such as arable crops). Examples of plant expression vectors comprise those which are described in detail in: Becker, D., Kemper, E., Schell, J., and Masterson , R. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol. 20:1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press, 1993, p. 15-38.
A plant expression cassette preferably comprises regulatory sequences which are capable of controlling the gene expression in plant cells and which are functionally linked so that each sequence can fulfill its function, such as transcriptional termination, for example polyadenylation signals. Preferred polyadenylation signals are those which are derived from Agrobacterium tumefaciens T-DNA, such as the gene 3 of the Ti plasmid pTiACH5, which is known as octoµl ne synthase (Gielen et al., EMBO J. 3 (1984) 835 et seq.) or functional equivalents of these, but all other terminators which are functionally active in plants are also suitable.
Since plant gene expression is very often not limited to transcriptional levels, a plant expression cassette preferably comprises other functionally linked sequences such as translation enhancers, for example the overdrive sequence, which comprises the 5'-untranslated tobacco mosaic virus leader sequence, which increases the protein/RNA ratio (Gallie et al., 1987, Nucl. Acids Research 15:8693-8711).
As described above, plant gene expression must be functionally linked to a suitable promoter which performs the expression of the gene in a timely, cell-specific or tissue-specific manner. Promoters which can be used are constitutive promoters (Benfey et al., EMBO J. 8 (1989) 2195-2202) such as those which are derived from plant viruses such as 35S CAMV (Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see also US 5352605 and WO 84/02913) or plant promoters such as the promoter of the Rubisco small subunit, which is described in US 4,962,028.

Other preferred sequences for the use in functional linkage in plant gene expression cassettes are targeting sequences which are required for targeting the gene product into its relevant cell compartment (for a review, see Kermode, Crit. Rev. Plant Sci. 15, 4 (1996) 285-423 and references cited therein), for example into the vacuole, the nucleus, all types of plastids, such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, the mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes and other compartments of plant cells.
As described above, plant gene expression can also be facilitated via a chemically inducible promoter (for a review, see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89-108). Chemically inducible promoters are particularly suitable if it is desired that genes are expressed in a time-specific manner. Examples of such promoters are a salicylic-acid-inducible promoter (WO 95/19443), a tetracyclin-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404) and an ethanol-inducible promoter.
Promoters which respond to biotic or abiotic stress conditions are also suitable promoters, for example the pathogen-inducible PRP1-gene promoter (Ward et al., Plant Mol. Biol. 22 (1993) 361-366), the heat-inducible hsp80 promoter from tomato (US 5,187,267), the cold-inducible alpha-amylase promoter from potato (WO 96/12814) or the wound-inducible µl nll promoter (EP-A-0 375 091).
The promoters which are especially preferred are those which bring about the expression of genes in tissues and organs in which fatty acid, liµl d and oil biosynthesis takes place, in seed cells such as the cells of endosperm and of the develoµl ng embryo. Suitable promoters are the naµl n gene promoters from oilseed rape (US 5,608,152), the USP promoter from Vicia faba (Baeuµlein et al., Mol. Gen. Genet, 1991, 225 (3):459-67), the oleosin promoter from Arabidopsis (WO 98/45461), the phaseolin promoter from Phaseolus vulgaris (US 5,504,200), the Bce4 promoter from Brassica (WO 91/13980) or the legumin B4 promoter (LeB4; Baeuµlein et al., 1992, Plant Journal, 2 (2):233-9), and promoters which bring about the seed-specific expression in monocotyledonous plants such as maize, barley, wheat, rye, rice and the like. Suitable promoters to be taken into consideration are the Ipt2 or Ipt1 gene promoter from barley (WO 95/15389 and WO 95/23230) or those which are described in WO 99/16890 (promoters from the barley hordein gene, the rice glutelin gene, the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene, the rye secalin gene).

In particular, the multiparallel expression of the ∆12-desaturases, ω3-desaturases, ∆9-elongases, ∆6-desaturases, ∆8-desaturases, ∆6-elongases, ∆5-desaturases, ∆5-elongases and/or ∆4-desaturases may be desired. Such expression cassettes can be introduced via a simultaneous transformation of a plurality of individual expression constructs or, preferably, by combining a plurality of expression cassettes on one construct. Also, it is possible to transform a plurality of vectors with in each case a plurality of expression cassettes and to transfer them to the host cell.
Likewise especially suitable are promoters which bring about the plastid-specific expression since plastids are the compartment in which the precursors and some end products of liµl d biosynthesis are synthetized. Suitable promoters such as the viral RNA-polymerase promoter, are described in WO 95/16783 and WO 97/06250, and the dpP promoter from Arabidopsis, described in WO 99/46394.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. The terms "transformation" and "transfection", conjugation and transduction, as used in the present context, are intended to comprise a multiplicity of prior-art processes for introducing foreign nucleic acid (for example DNA) into a host cell, including calcium phosphate or calcium chloride copreciµl tation, DEAE-dextran-rnediated transfection, lipofection, natural competence, chemically mediated transfer, electroporation or particle bombardment. Suitable methods for the transformation or transfection of host cells, including plant cells, can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals, such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, Ed.: Gartland and Davey, Humana Press, Totowa, New Jersey.
The host organisms which are advantageously used are plant cells, preferably plants or parts thereof. Especially preferred plants are plants such as oilseed plants or oil crops, which comprise large amounts of liµl d compounds, such as oilseed rape, evening primrose, hemp, thistle, peanut, canola, linseed, soybean, safflower, Indian mustard, sunflower, borage or plants such as maize, wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper, Tagetes, Solanaceae plants such as potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees (oil pal.,, coconut) and perennial grasses and fodder crops. Especially preferred plants according to the invention are oil crops such as soybean,

NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 183, SEQ ID NO: 193, SEQ ID NO: 197, SEQ ID NO: 199 or SEQ ID NO: 201. The homology was calculated over the entire amino acid or nucleic acid sequence region. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences. In this context, the algorithms of Needieman and Wunsch or Smith and Waterman give particularly reliable results. To carry out the sequence alignments, the program µl leUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 (1989: 151-153) or the programs Gap and BestFit [Needieman and Wunsch (J. Mol. Biol. 48; 443-453 (1970) and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981)], which are part of the GCG software packet [Genetics Computer Group, 575 Science Drive, Madison Wisconsin, USA 53711 (1991)], were used. The sequence homology values detailed above in percent were determined using the program GAP over the entire sequence region with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which, unless otherwise specified, were always used as standard settings for sequence alignments.
Homologs of the abovementioned nucleic acid sequences also mean for example bacterial, fungal and plant homologs, truncated sequences, single-stranded DNA or RNA of the coding and noncoding DNA sequence or else derivatives such as, for example, promoter variants. The promoters upstream of the nucleotide sequences stated can be modified by one or more nucleotide substitutions, by insertion(s) and/or deletion(s), without, however, the functionality or activity of the promoters being adversely affected. Furthermore, it is possible that the activity of the promoters is increased by modifying their sequence, or that they are replaced completely by more active promoters, including those from heterologous organisms.
The abovementioned nucleic acids and protein molecules with ∆12-desaturase, ω3--desaturase, ∆9-elongase, ∆6-desaturase, ∆8-desaturase, ∆6-elongase, ∆5-desaturase, ∆5-elongase and/or ∆4-desaturase activity which are involved in the metabolism of iiµl ds and fatty acids, PUFA cofactors and enzymes or in the transport of lipophilic compounds across membranes are used in the process according to the invention for modulating the production of PUFAs in transgenic plants such as maize, wheat, rye, oats, triticale, rice, barley, soybean, peanut, cotton, Linum species such as linseed or flax, Brassica species such as oilseed rape, canola, Indian mustard and

turnip rape, pepper, sunflower, borage, evening primrose and Tagetes, Solanaceae plants such as potato, tobacco, eggplant or tomato, Vicia species, pea, cassava, alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees (oil pal.,, coconut) and perennial grasses and fodder crops either directly (for example when the overexpression or optimization of a fatty acid biosynthetic protein has a direct effect on the yield, production and/or production efficiency of the fatty acid from modified organisms) and/or can have an indirect effect which nevertheless entails an increase in the yield, production and/or production efficiency of the PUFAs or a decrease of undesired compounds (for example when the modulation of the metabolism of liµl ds and fatty acids, cofactors and enzymes results in changes in the yield, production and/or production efficiency or the composition of the desired compounds within the cells which, in turn, can have an effect on the production of one or more fatty acids).
Brassicaceae, Boraginaceae, Primulaceae or Linaceae are especially suitable for the production of PUFAs, preferably of arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid. Especially suitable for the production of PUFAs with the nucleic acid sequences according to the invention, advantageously, as described, in combination with further desaturases and elongases are Indian mustard (Brassica juncea), oilseed rape and Camelina sativa.
The combination of a variety of precursor molecules and biosynthetic enzymes leads to the production of different fatty acid molecules, which has a major effect on the composition of the iiµl ds since polyunsaturated fatty acids (= PUFAs) are incorporated not only into triacylglycerol but also into membrane liµl ds.
Brassicaceae, Boraginaceae, Primulaceae or Linaceae are especially suitable for the production of PUFAs, for example stearidonic acid, eicosapentaenoic acid or docosahexaenoic acid. Linseed (Linum usitatissumum) and Brassica juncea and Camelina sativa are especially advantageously suitable for the production of PUFAs with the nucleic acid sequences according to the invention, advantageously, as described, in combination with further desaturates and elongases.
Liµl d synthesis can be divided into two sections: the synthesis of fatty acids and their binding to sn-glycerol-3-phosphate, and the addition or modification of a polar head group. Usual liµl ds which are used in membranes comprise phospholiµl ds, glycoliµl ds, sphingoliµl ds and phosphoglycerides. Fatty acid synthesis starts with the conversion of acetyl-CoA into malonyl-CoA by acetyl-CoA carboxylase or into acetyl-ACP by acetyl transacylase. After condensation reaction, these two product molecules together form

acetoacetyl-ACP, which is converted via a series of condensation, reduction and dehydration reactions so that a saturated fatty acid molecule with the desired chain length is obtained. The production of the unsaturated fatty acids from these molecules is catalyzed by specific desaturases, either aerobically by means of molecular oxygen or anaerobically (regarding the fatty acid synthesis in microorganisms, see F.C. Neidhardt et al. (1996) E. coli and Sal.,onella. ASM Press: Washington, D.C., p. 612-636 and references cited therein; Lengeler et al. (Ed.) (1999) Biology of Procaryotes. Thieme: Stuttgart, New York, and the references therein, and Magnuson, K., et al. (1993) Microbiological Reviews 57:522-542 and the references therein). To undergo the further elongation steps, the resulting phospholiµl d-bound fatty acids must be returned from the phospholiµl ds to the fatty acid CoA ester pool. This is made possible by acyl-CoA:lysophospholiµl d acyltransferases. Moreover, these enzymes are capable of transferring the elongated fatty acids from the CoA esters back to the phospholiµl ds. If appropriate, this reaction sequence can be followed repeatedly.
Examples of precursors for PUFA biosynthesis are oleic acid, linoleic acid and linolenic acid. These C18-carbon fatty acids must be elongated to C20 and C22 to obtain fatty acids of the eicosa and docosa chain type. It is possible, with the aid of the desaturases used in the process, such as the ∆12-, ω3-, ∆4-, ∆5-, ∆6- and ∆8-desaturases and/or the ∆5-, ∆6-, ∆9-elongases to produce arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid, advantageously eicosapentaenoic acid and/or docosahexaenoic acid, and subsequently to use them for a variety of purposes in applications in the fields of foodstuffs, feedstuffs, cosmetics or Pharmaceuticals. Using the abovementioned enzymes, C20- and/or C22-fatty acids with at least two, advantageously at least three, four, five or six double bonds in the fatty acid molecule, preferably C20- or C22-fatty acids with advantageously four, five or six double bonds in the fatty acid molecule can be produced. The desaturation can take place before or after elongation of the fatty acid in question. This is why the products of the desaturase activities and the further possible desaturation and elongation lead to preferred PUFAs with a higher degree of desaturation, including a further elongation of C20- to C22-fatty acids, to fatty acids such as y-linolenic acid, dihomo-y-linolenic acid, arachidonic acid, stearidonic acid, eicosatetraenoic acid or eicosapentaenoic acid. Substrates of the desaturases and elongases used in the process according to the invention are C16-, C18- or C20-fatty acids such as, for example, linoleic acid, y-linolenic acid, a-linolenic acid, dihomo-y-linolenic acid, eicosatetraenoic acid or stearidonic acid. Preferred substrates are linoleic acid, y-linolenic acid and/or a-linolenic acid, dihomo-y-linolenic acid or arachidonic acid, eicosatetraenoic acid or eicosapentaenoic acid. The synthesized C20-

to C22-fatty acids with at least two, three, four, five or six, advantageously at least four, five or six double bonds in the fatty acid are obtained in the process according to the invention in the form of the free fatty acid or in the form of its esters, for example in the form of its glycerides.
The term "glyceride" is understood as meaning glycerol esterified with one, two or three carboxyl radicals (mono-, di- ortriglyceride). "Glyceride" is also understood as meaning a mixture of various glycerides. The glyceride or glyceride mixture can comprise further additions, for example free fatty acids, antioxidants, proteins, carbohydrates, vitamins and/or other substances.
A"glyceride" for the purposes of the process according to the invention is furthermore understood as meaning derivatives which are derived from glycerol. In addition to the above-described fatty acid glycerides, these also include glycerophospholiµl ds and glyceroglycoliµl ds. Preferred examples which may be mentioned here are the glycerophospholiµl ds such as lecithin (phosphatidylcholine), cardioliµl n, phosphatidylglycerol, phosphatidylserine and alkylacylglycerophospholiµl ds.
Furthermore, fatty acids must subsequently be transported to various sites of modification and incorporated into the triacylglycerol storage liµl d. A further important step in liµl d synthesis is the transfer of fatty acids onto the polar head groups, for example by glycerol-fatty-acid acyltransferase (see Frentzen, 1998, Liµl d, 100(4-5):161-166).
Publications on plant fatty acid biosynthesis, desaturation, the liµl d metabolism and the transmembrane transport of fatty compounds, beta-oxidation, fatty acid modification * and cofactors, triacylglycerol storage and assembly, including the references therein, see the following articles: Kinney, 1997, Genetic Engineering, Ed., JK Setlow, 19:149-166; Ohlrogge and Browse, 1995, Plant Cell 7:957-970; Shanklin and Cahoon, 1998, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:611-641; Voelker, 1996, Genetic Engineering, Ed.: JK Setlow, 18:111-13; Gerhardt, 1992, Prog. Liµl d R. 31:397-417; Guhnemann-Schafer & Kindl, 1995, Biochim. Biophys Acta 1256:181-186; Kunau et al., 1995, Prog. Liµl d Res. 34:267-342; Stymme et al., 1993, in: Biochemistry and Molecular Biology of Membrane and Storage Liµl ds of Plants, Ed.: Murata and Somerville, Rockville, American Society of Plant Physiologists, 150-158, Murphy & Ross 1998, Plant Journal. 13(1):1-16.
The PUFAs produced in the process comprise a group of molecules which higher

animals are no longer capable of synthesizing and must therefore take up, or which higher animals are no longer capable of synthesizing themselves in sufficient quantity and must therefore take up additionally, although they can be readily synthesized by other organisms such as bacteria; for example, cats are no longer capable of synthesizing arachidonic acid.
Phospholiµl ds are to be understood as meaning, for the purposes of the invention, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and/or phosphatidylinositol, advantageously phosphatidylcholine.
The terms "production" or "productivity" are known in the art and refer to the concentration of the fermentation product (compounds of the formula I) formed within a certain period of time and a certain fermentation volume (for example kg of product per hour per liter). They also encompass the productivity within a plant cell or a plant, i.e. the content of the desired fatty acids produced in the process based on the content of all fatty acids in this cell or plant. The term production efficiency encompasses the time required for obtaining a certain amount of product (for example the time required by the cell for establishing a certain throughput rate of a fine chemical). The term "yield" or "product/carbon yield" is known in the art and comprises the efficiency of the conversion of the carbon source into the product (i.e. the fine chemical). This is usually expressed for example as kg of product per kg of carbon source. By increasing the yield or production of the compound, the amount of the obtained molecules or of the suitable obtained molecules of this compound in a certain amount of culture is increased over a specified period.
The terms "biosynthesis" or "biosynthetic pathway" are known in the art and comprise the synthesis of a compound, preferably of an organic compound, by a cell starting from intermediates, for example in a multistep process which is highly regulated. The terms "catabolism" or "catabolic pathway" are known in the art and comprise the cleavage of a compound, preferably of an organic compound, by a cell to give catabolytes (in more general terms, smaller or less complex molecules), for example in a multistep process which is highly regulated.
The term "metabolism" is known in the art and encompasses the totality of the biochemical reactions which take place in an organism. Thus, the metabolism of a certain compound (for example the metabolism of a fatty acid) comprises the totality of the biosynthetic, modification and catabolic pathways of this compound in the cell.

This invention is illustrated in greater detail by the examples which follow, which are not to be construed as limiting. The content of all of the references, patent applications, patents and published patent applications cited in the present patent application is herewith incorporated by reference.
Examples
Example 1: General cloning methods:
The cloning methods such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linkage of DNA fragments, transformation of £ coli cells, bacterial cultures and the sequence analysis of recombinant DNA were carried out as described by Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6).
Example 2: Sequence analysis of recombinant DNA:
Recombinant DNA molecules were sequenced with an ABI laser fluorescence DNA sequencer by the process of Sanger (Sanger et al. (1977) Proc. Natl. Acad. Sci. US∆74, 5463-5467). Fragments resulting from a polymerase chain reaction were sequenced and verified to avoid polymerase errors in constructs to be expressed.
Example 3: Cloning genes from Oncorhynchus mykiss
As the result of a search for conserved regions in the protein sequences corresponding to the elongase genes detailed in the application, two sequences with suitable motifs were identified in the Genbank sequence database.

Total RNAfrom Oncorhynchus mykiss was isolated with the aid of the RNAeasy Kit from Qiagen (Valencia, CA, US). Poly-A+ RNA (mRNA) was isolated from the total RNA with the aid of oligo-dT cellulose (Sambrook et al., 1989). The RNA was subjected to reverse transcription using the reverse transcription system kit from Promega, and

the cDNA synthesized was cloned into the lambda ZAP vector (lambda ZAP Gold, Stratagene). The cDNA was depackaged in accordance with the manufacturer's instructions to give the plasmid DNA. The cDNA plasmid library was then used for the PCR for cloning expression plasmids.
Example 4: Cloning expression plasmids for the heterologous expression in yeasts
To clone the two sequences for heterologous expression in yeasts, the following oligonucleotides were used for the PCR reaction:

Composition of the PCR mix (50 µl):
5.00µl template cDNA
5.00 µl 10X buffer (Advantage polymerase) + 25mM MgCI2
5.00 µl of2mMdNTP
1.25 µl of each primer (10 pmol/µl
0.50 µl of Advantage polymerase (Clontech)
PCR reaction conditions: Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35
The PCR product was first incubated for 2 hours at 37°C with the restriction enzymes Hindlll and BamHI. The yeast expression vector pYES3 (Invitrogen) was incubated in the same manner. Thereafter, the 812 bp PCR product and the 905 bp PCR product and the vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit following the manufacturer's instructions. Thereafter, the vector and the elongase cDNA were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmids pYES3-OmELO2 and pYES3-OmELO3 were verified by sequencing and transformed into the Saccharomyces strain INVSd (Invitrogen) by

from Roche was used for this purpose. The resulting plasmids pSUN-OmELO2 and pSUN-OmELO3 were verified by sequencing.
pSUN300 is a derivative of the plasmid pPZP (Hajdukiewicz P., Svab, Z, Maliga P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25:989-994). pSUN-USP originated from pSUN300 by inserting a USP promoter as EcoRI fragment into pSUN 300. The polyadenylation signal is that of the octoµl n synthase gene from the A. tumefaciens Ti plasmid (ocs terminator, Genbank Accession V00088) (De Greve, H., Dhaese, P., Seurinck, J., Lemmers, M., Van Montagu, M. and Schell, J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octoµl ne synthase gene J. Mol. Appl. Genet. 1 (6), 499-511 (1982). The USP promoter corresponds to the nucleotides 1-684 (Genbank Accession X56240), part of the noncoding region of the USP gene being present in the promoter. The promoter fragment, which is 684 base pairs in size, was amplified via a PCR reaction by standard methods, by means of commercially available T7 standard primer (Stratagene) and with the aid of a synthetized primer (primer sequence:
5'-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTTGGCTATGAA-3\ SEQ ID NO: 174). The PCR fragment was recut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator. This gave rise to the plasmid named pSUN-USP. The construct was used for transforming Arabidopsis thaliana, oilseed rape, tobacco and linseed.
Example 6: Liµl d extraction from yeasts and seeds:
The effect of the genetic modification in plants, fungi, algae, ciliates or on the production of a desired compound (such as a fatty acid) can be determined by growing the modified microorganisms or the modified plant under suitable conditions (such as those described above) and analyzing the medium and/or the cellular components for the elevated production of the desired product (i.e. of the liµl ds or a fatty acid). These analytical techniques are known to the skilled worker and comprise spectroscopy, thin-layer chromatography, various types of staining methods, enzymatic and microbiological methods and analytical chromatography such as high-performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987) "Applications of HPLC in Biochemistry" in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol. 3, Chapter III: "Product recovery and purification", p. 469-714, VCH: Weinheim; Belter, P.A., et al.

(1988) Bioseparations: downstream processing for Biotechnology, John Wiley and Sons; Kennedy, J.F., and Cabral, J.M.S. (1992) Recovery processes for biological Materials, John Wiley and Sons; Shaeiwitz, JA, and Henry, J.D. (1988) Biochemical Separations, in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3; Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, FJ. (1989) Separation and purification techniques in biotechnology, Noyes Publications).
In addition to the abovementioned methods, plant liµl ds are extracted from plant material as described by Cahoon et al. (1999) Proc. Natl. Acad. Sci. USA 96 (22):12935-12940 and Browse et al. (1986) Analytic Biochemistry 152:141-145. The qualitative and quantitative analysis of liµl ds or fatty acids is described by Christie, William W., Advances in Liµl d Methodology, Ayr/Scotland: Oily Press (Oily Press Liµl d Library; 2); Christie, William W., Gas Chromatography and Liµl ds. A Practical Guide -Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307 pp. (Oily Press Liµl d Library; 1); "Progress in Liµl d Research, Oxford: Pergamon Press, 1 (1952) -16 (1977) under the title: Progress in the Chemistry of Fats and Other Liµl ds CODEN.
In addition to measuring the end product of the fermentation, it is also possible to analyze other components of the metabolic pathways which are used for the production of the desired compound, such as intermediates and by-products, in order to determine the overall production efficiency of the compound. The analytical methods comprise measuring the amount of nutrients in the medium (for example sugars, hydrocarbons, nitrogen sources, phosphate and other ions), measuring the biomass composition and the growth, analyzing the production of conventional metabolytes of biosynthetic pathways and measuring gases which are generated during the fermentation. Standard methods for these measurements are described in Applied Microbial Physiology; A Practical Approach, P.M. Rhodes and P.F. Stanbury, Ed., IRL Press, p. 103-129; 131-163 and 165-192 (ISBN: 0199635773) and references cited therein.
One example is the analysis of fatty acids (abbreviations: FAME, fatty acid methyl ester; GC-MS, gas liquid chromatography/mass spectrometry; TAG, triacylglycerol; TLC, thin-layer chromatography).
The unambiguous detection for the presence of fatty acid products can be obtained by analyzing recombinant organisms using analytical standard methods: GC, GC-MS or TLC, as described on several occasions by Christie and the references therein (1997, in: Advances on Liµl d Methodology, Fourth Edition: Christie, Oily Press, Dundee, 119-

169; 1998, Gaschromatographie-Massenspektrometrie-Verfahren [Gas chromatography/mass spectrometric methods], Liµl de 33:343-353).
The material to be analyzed can be disrupted by sonication, grinding in a glass mill, liquid nitrogen and grinding or via other applicable methods. After disruption, the material must be centrifuged. The sediment is resuspended in distilled water, heated for 10 minutes at 100°C, cooled on ice and recentrifuged, followed by extraction for one hour at 90°C in 0.5 M sulfuric acid in methanol with 2% dimethoxypropane, which leads to hydrolyzed oil and liµl d compounds, which give transmethylated liµl ds. These fatty acid methyl esters are extracted in petroleum ether and finally subjected to a GC analysis using a caµl llary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) at a temperature gradient of between 170°C and 240°C for 20 minutes and 5 minutes at 240°C. The identity of the resulting fatty acid methyl esters must be defined using standards which are available from commercial sources (i.e. Sigma).
Plant material is initially homogenized mechanically by comminuting in a pestle and mortar to make it more amenable to extraction.
This is followed by heating at 100°C for 10 minutes and, after cooling on ice, by resedimentation. The cell sediment is hydrolyzed for one hour at 90°C with 1 M methanolic sulfuric acid and 2% dimethoxypropane, and the liµl ds are transmethylated. The resulting fatty acid methyl esters (FAMEs) are extracted in petroleum ether. The extracted FAMEs are analyzed by gas liquid chromatography using a caµl llary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) and a temperature gradient of from 170°C to 240°C in 20 minutes and 5 minutes at 240°C. The identity of the fatty acid methyl esters is confirmed by comparison with corresponding FAME standards (Sigma). The identity and position of the double bond can be analyzed further by suitable chemical derivatization of the FAME mixtures, for example to give 4,4-dimethoxyoxazoIin derivatives (Christie, 1998) by means of GC-MS..
Yeasts which had been transformed with the plasmids pYES3, pYES3~OmELO2 and pYES3-OmELO3 as described in Example 4 were analyzed as follows:
The yeast cells from the main cultures were harvested by centrifugation (100 x g, 10 min, 20°C) and washed with 100 mM NaHCO3, pH 8.0 in order to remove residual medium and fatty acids. Fatty acid methyl esters (FAMEs) were prepared with the yeast cell sediments by acid methanolysis. To this end, the cell sediments were incubated for 1 hour at 80°C with 2 m) of 1N methanolic suifuric acid and 2% (v/v)

dimethoxypropane. The FAMEs were extracted by twice extracting with petroleum ether (PE). To remove non-derivatized fatty acids, the organic phases were washed in each case once with 2 µl of 100 mM NaHCO3, pH 8.0, and 2 µl of distilled water. Thereafter, the PE phases were dried with Na2SO4, evaporated under argon and taken up in 100 µl of PE. The samples were separated on a DB-23 caµl llary column (30 m, 0.25 mm, 0.25 µm, Agilent) in a Hewlett-Packard 6850 gas chromatograph with flame ionization detector. The conditions for the GLC analysis were as follows: the oven temperature was programmed from 50°C to 250°C with an increment of 5°C/min and finally 10 minutes at 250°C (holding).
The signals were identified by comparing the retention times with corresponding fatty acid standards (Sigma).
The methodology is described for example in Naµl er and Michaelson, 2001, Liµl ds 36(8):761-766; Sayanova et al., 2001, Journal of Experimental Botany, 52(360):1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218.
Example 7: Functional characterization of OmELO2 and OmELO3:
OmELO2 shows no elongase activity, while a pronounced activity was detected for OmELO3, using different substrates. The substrate specificity of OmElo3 was determined after expression and feeding with various fatty acids (Figure 2). The fed substrates can be detected in large amounts in all transgenic yeasts. All transgenic yeasts show that new fatty acids have been synthesized, to the products of the OmElo3 reaction. This means that the gene OmEio3 was expressed functionally.
Figure 2 demonstrates that OmElo3 has a substrate specificity which leads to the elongation of ∆5- and ∆6-fatty acids with one w-double bond with high specificity. Moreover, ω6--fatty acids (C18 and C20) were also elongated, with less specificity. The best substrates for OmElo3 were stearidonic acid (C18:4 ω3-) and eicosapentaenoic acid (C20:5 ω3-) (up to 66% elongation).
Example 8: Reconstitution of the synthesis of DHA in yeast
The reconstitution of the biosynthesis of DHA (22:6 ω3-) was carried out starting from EPA (20:5 ω3-) or stearidonic acid (18:4 ω3-) by coexpressing OmElo3 together with the Euglena gracilis ∆4-desaturase or the Phaeodactylum tricornutum ∆5-desaturase and

the Euglena gracilis ∆4-clesaturase. To this end, the expression vectors pYes2-EgD4 and pESCLeu-PtD5 were additionally constructed. The abovementioned yeast strain which is already transformed with pYes3~OmElo3 (SEQ ID NO: 55), was then transformed further with pYes2-EgD4, or simultaneously with pYes2-EgD4 and pESCLeu-PtD5. The transformed yeasts were selected on complete minimal dropout tryptophan and uracil medium agar plates supplemented with 2% glucose in the case of the pYes3-pYes3-OmEIO/pYes2-EgD4 strain and complete minimal dropout tryptophan, uracil and leucine medium in the case of the pYes3-OmEIO/pYes2-EgD4+pESCLeu-RD5 strain. Expression was then induced by addition of 2% (w/v) galactose. The cultures were subsequently incubated for a further 120 hours at 15°C.
Figure 3 shows the fatty acid profiles of transgenic yeasts which have been fed 20:5 ω3. In the control yeast (A), which had been transformed with the vector pYes3-OmElo3 and the blank vector pYes2, 20:5 Ω3 was elongated highly efficiently to give 22:5 ω3 (65% elongation). The additional introduction of the EEg∆4-desaturase led to the conversion of 22:5 ω3 into 22:6 ω3 DHA. The fatty acid composition of the transgenic yeasts is shown in Figure 5. After coexpression of OmElo3 and EgD4, up to 3% DHA was detected in yeasts.
In a further coexpression experiment, OmElo3, EgD4 and a ∆5-desaturase from P. tricomutum (PtD5) were expressed together. The transgenic yeasts were fed stearidonic acid (18:4 ω3) and analyzed (Figure 4). The fatty acid composition of these yeasts is shown in Figure 5. OmElo3 elongated the fed fatty acid 18:4 ω3- to give 20:4 ω3- (60% elongation). The latter was desaturated by PtD5 to give 20:5 ω3-. The PtD5 activity amounted to 15%. Furthermore, 20:5 ω3 was elongated by EmElo3 to give 22:5 to3. Thereafter, the newly synthesized 22:5 co3 was desaturated to give 22:6 OJ3 (DHA). Up to 0.7% of DHA was obtained in these experiments.
These experiments demonstrate that the sequences OmElo3, EgD4 and PtD5 which are used in the present invention are suitable for the production of DHA in eukaryotic cells.
Example 9: Generation of transgenic plants
a) Generation of transgenic oilseed rape plants (modified process of Moloney et al., 1992, Plant Cell Reports, 8:238-242)

The binary vectors in Agrobacterium tumefaciens C58C1 :pGV2260 or Escherichia coli (Deblaere et al, 1984, Nucl. Acids. Res. 13, 4777-4788) can be used for generating transgenic oilseed rape plants. To transform oilseed rape plants (Var. Drakkar, NPZ Nordeutsche Pflanzenzucht, Hohenlieth, Germany), a 1:50 dilution of an overnight culture of a positively transformed agrobacterial colony in Murashige-Skoog medium (Murashige and Skoog 1962 Physioi. Plant. 15, 473) supplemented with 3% sucrose (3MS medium) is used. Petiols or hypocotyls of freshly germinated sterile oilseed rape plants (in each case approx. 1 cm2) are incubated with a 1:50 agrobacterial dilution for 5-10 minutes in a petri dish. This is followed by 3 days of coincubation in the dark at 25°C on 3MS medium supplemented with 0.8% Bacto agar. The cultures are then grown for 3 days at 16 hours light/8 hours dark. The cultivation is then continued in a weekly rhythm on MS medium supplemented with 500 mg/l Claforan (cefotaxim sodium), 50 mg/l kanamycin, 20 p,M benzylaminopurine (BAP), now supplemented with 1.6 g/l of glucose. Growing shoots are transferred to MS medium supplemented with 2% sucrose, 250 mg/l Claforan and 0.8% Bacto agar. If no roots have developed after three weeks, 2-indoiebutyric acid is added to the medium as growth hormone for rooting.
Regenerated shoots were obtained on 2MS medium supplemented with kanamycin and Claforan; after rooting, they were transferred to compost and, after growing on for two weeks in a controlled-environment cabinet or in the greenhouse, allowed to flower, and mature seeds were harvested and analyzed by liµl d analysis for elongase expression, such as ∆5-elongase or ∆6-elongase activity. In this manner, lines with elevated contents of polyunsaturated C20- and C2rfetty acids can be identified.
b) Generation of transgenic linseed plants
Transgenic linseed plants can be generated for example by the process of Bell et al., 1999, In Vitro Cell. Dev. Biol.-Plant. 35(6):456-465 by means of particle bombardment. Usually, an agrobacteria-mediated transformations was used for the transformation of linseed, for example by the process of µlynarova et al. (1994), Plant Cell Report 13: 282-285.
Example 10: Cloning ∆5-elongase genes from Thraustochytrium aureum ATCC34304 and Thraustochytrium ssp.
Comparisons of the various elongase protein sequences found in the present application enabled the definition of conserved nucleic add regions (histidin box: His-

Val-X-His-His, tyrosin box: Met-Tyr-X-Tyr-Tyr). An EST database of T. aureum ATCC34304 and Thraustochytrium ssp. was screened for further ∆5-elongases with the aid of these sequences. The following new sequences were found:

Total RNA from T. aureum ATCC34304 and Thraustochytrium ssp. was isolated with the aid of the RNAeasy Kits from Qiagen (Valencia, CA, US). mRNA was isolated from the total RNA with the aid of the poIyATract isolation system (Promega). The mRNA was subjected to reverse transcription using the Marathon cDNA Amplification Kit (BD Biosciences) and adaptors were ligated in accordance with the manufacturer's instructions. The cDNA library was then employed for the PCR for cloning expression µl asmids by means of 5'- and 3'-RACE (raµl d amplification of cDNA ends).
Example 11: Cloning expression µl asmids for the heterologous expression in yeasts
To clone the sequence for heterologous expression in yeasts, the following
oligonucleotides were used for the PCR reaction:

Elongation temperature: 2 min 72°C Number of cycles: 35
The PCR products BioTaurELOl (see (SEQ ID NO: 65) and TL16y2 (see SEQ ID NO: 83) were incubated for 30 minutes at 21 °C with the yeast expression vector pYES2.1-TOPO (Invitrogen) following the manufacturer's instructions. The PCR product is ligated into the vector by means of a T overhang and activity of a topoisomerase (Invitrogen). After incubation, E. coli DH5a cells were transformed. Suitable clones were identified by PCR, the plasmid DNA was isolated by means of Qiagen DNAeasy Kit and verified by sequencing. The correct sequence was then transformed into the Saccharomyces strain INVSd (Invitrogen) by electroporation (1500 V). As a control, the blank vector pYES2.1 was transformed in parallel. The yeasts were subsequently plated onto complete uracil dropout minimal medium supplemented with 2% glucose. Cells which were capable of growing in the medium without uracil thus comprise the corresponding plasmids pYES2.1, pYES2.1-BioTaurEL01 and pYES2.1-TL16y2. After the selection, in each case two transformants were selected for further functional expression.
Example 12: Cloning expression plasmids for the seed specific expression in plants
A further transformation vector based on pSUN-USP was generated for the transformation of plants. To this end, Notl cleavage sites were introduced at the 5' and 3' termini of the coding sequence, using the following primer pair:
PSUN-BioTaurEL01
Forward: 5'-GCGGCCGCATAATGACGAGCAACATGAGC (SEQ ID NO: 166)
Reverse: 3J-GCGGCCGCTTAGGCCGACTTGGCCTTGGG (SEQ ID NO: 167)
PSUN-TL16y2
Forward: 5'-GCGGCCGCACCATGGACGTCGTCGAGCAGCAATG (SEQ ID NO: 168)
Reverse: 5'-GCGGCCGCTTAGATGGTCTTCTGCTTCTTGGGCGCC (SEQ ID NO:
169)
Composition of the PCR mix (50 µl):
5.00 µl template cDNA
5.00 µl 10x buffer (Advantage polymerase)+ 25mM MgCI2
5.00 µl 2mM dNTP
1.25 µl of each primer (10 pmol/ µl)

0.50 µl Advantage polymerase
The Advantage polymerase from Clontech was employed.
PCR reaction conditions:
Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35
The PCR products were incubated with the restriction enzyme Notl for 16 hours at 37°C. The plant expression vector pSUN300-USP was incubated in the same manner. Thereafter, the PCR products and the 7624 bp vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit following the manufacturer's instructions. Thereafter, vector and PCR products were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmids pSUN-BioTaurEL01 and pSUN-TL16y2 were verified by sequencing.
pSUN300 is a derivative of plasmid pPZP (Hajdukiewicz,P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989-994). pSUN-USP originated from pSUN300, by inserting a USP promoter into pSUN300 in the form of an EcoRI fragment. The polyadenylation signal is that of the octoµl ne synthase gene from the A. tumefaciens Ti plasmid (ocs-Terminator, Genbank Accession V00088) (De Greve,H., Dhaese,P., Seurinck,J., Lemrners,M., Van Montagu,M. and Schell,J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octoµl ne synthase gene J. Mol. Appl. Genet. 1 (6), 499-511 (1982)). The USP promoter corresponds to nucleotides 1 to 684 (Genbank Accession X56240), where part of the noncoding region of the USP gene is present in the promoter. The promoter fragment which is 684 base pairs in size was amplified by a PCR reaction and standard methods with the aid of a synthesized primer and by means of a commercially available T7 standard primer (Stratagene). (Primer sequence:
5'-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-3', SEQ ID NO: 165). The PCR fragment was recut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator. This gave rise

to the plasmid with the name pSUN-USP. The construct was used for the transformation of Arabidopsis thaliana, oilseed rape, tobacco and linseed.
Liµl ds were extracted from yeasts and seeds as described for Example 6. Example 13: Functional characterization of BioTaurELOl and TL16y2:
The substrate specificity of BioTaurELOl was determined after expression and feeding of various fatty acids (Figure 6). Figure 6 shows the feeding experiments for determining the functionality and substrate specificity with yeast strains comprising either the vector pYes2.1 (control) or the vector pYes2.1 -BioTaurELOl (= BioTaur) with the ∆5-elongase. In both approaches, 200 ^m of y-linolenic acid and eicosapentaenoic acid were added to the yeast incubation medium and incubated for 24 hours. After the fatty acids had been extracted from the yeasts, they were transmethylated and separated by gas chromatography. The elongation products originating from the two fatty acids which had been fed are identified by arrows.
The substrates which had been fed can be detected in large amounts in all transgenic yeasts. All transgenic yeasts show that new fatty acids have been synthesized, the products of the BioTaurELOl reaction. This means that the gene BioTaurELOl has been expressed functionally.
Figure 6 shows that BioTaurELOl has a substrate specificity which leads with high specificity to the elongation of ∆5- and ∆6-fatty acids with one ω3-double bond. Moreover, ω6--fatty acids (C18 and C20) were also elongated. y-Linolenic acid (C18:3 ω6-) is converted with a conversion rate of 65.28%, stearidonic acid (C18:4 ω3) with a conversion rate of 65.66% and eicosapentaenoic acid (C20:5 0)3) with a conversion rate of 22.01%. The substrate specificities of the various feeding experiments are shown in Table 6 (see end of the description).
The conversion rate of GLA when feeding GLA and EPA was 65.28%. The conversion rate of EPA, again when feeding GUK and EPA, was 9.99%. When only EPA was fed, the EPA conversion rate was 22.01%. Arachidonic acid (= ARA) was also converted when fed. The conversion rate was 14.47%. Stearidonic acid (= SDA) was also converted. In this case, the conversion rate was 65.66%.
The functionality and substrate specificity of TL16y2 were determined after expression and feeding of various fatty acids. Table 7 shows the feeding experiments. The feeding

experiments were carried out in the same manner as described for BioTaurELOL The substrates which have been fed can be detected in large amounts in all transgenic yeasts. The transgenic yeasts demonstrated the synthesis of novel fatty acids, the products of the TL16y2 reaction (Fig. 11). This means that the gene TL16y2 has been expressed functionally.

The results with TL16y2, which are shown in Table 7, show the following conversion rates in % of the control: a) conversion rate of EPA in % (250 pm): 8%, b) conversion rate of EPA in % (50 jam): 41%; c) conversion rate of ARA in %: 20.3%, d) conversion rate of SDA in %: 79.4%, and e) conversion rate of GLA in %; 74.9%.

Thus, TL16y2 shows ∆5-, ∆6- and ∆8-elongase activity. The activity is highest for CIS-fatty acids with ∆6-double bond. Then, C20-fatty acids with a ∆5- or ∆8-double bond are elongated, depending on the concentration of fatty acids which are fed.
Example 14: Cloning genes from Ostreococcus tauri
The search for conserved regions in the protein sequences with the aid of the elongase genes with ∆5-elongase activity or ∆6-elongase activity which are shown in the application allowed the identification of sequences with suitable motifs in an Ostreococcus tauri sequence database (genomic sequences).
The sequences were the following:

OtEloi shows the highest similarity with an elongase from Danio rerio (GenBank AAN77156; identity approx. 26%), while OtElo2 shows the highest similarity with the Physcomitrella Elo (PSE) [approx. 36% identity] (aiignements were carried out using the tBLASTn algorithm (Altschul et al., J. Mol. Biol. 1990, 215: 403-410).
The cloning procedure was as follows:
40 µl of an Ostreococcus tauri culture in the stationary phase were spun down, resuspended in 100 p.l of double-distilled water and stored at -20°C. The respective genomic DNAs were amplified on the basis of the PCR process. The relevant primer pairs were selected in such a way that they bore the yeast consensus sequence for highly efficient translation (Kozak, Cell 1986, 44:283-292) next to the start codon. The amplification of the OtElo DNAs was carried out in each case using 1 \i\ of defrosted cells, 200 jim of dNTPs, 2.5 U Taq polymerase and 100 pmol of each primer in a total volume of 50 jj. The PCR conditions were as follows: first denaturation for 5 minutes at 95°C, followed by 30 cycles of 30 seconds at 94°C, 1 minute at 55°C and 2 minutes at 72°C, and a last elongation step of 10 minutes at 72°C.
Example 15: Cloning expression plasmids for the heterologous expression in yeasts: To characterize the function of the Ostreococcus tauri elongases, the open reading

frames of the DNAs in question were cloned downstream of the galactose-inducible GAL1 promoter of pYES2.1 A/5-His-TOPO (Invitrogen), giving rise to pOTE1 and pOTE2.
The Saccharomyces cerevisiae strain 334 was transformed by electroporation (1500 v) with the vector pOTE1 or pOTE2. A yeast which was transformed with the blank vector pYES2 was used as the control. The transformed yeasts were selected on complete minimal dropout uracil medium (CMdum) agar plates supplemented with 2% glucose. After the selection, in each case three transformants were selected for the further functional expression.
To express the Ot elongases, precultures of in each case 5 µl of dropout uracil CMdum liquid medium supplemented with 2% (w/v) raffinose were inoculated with the selected transformants and incubated for 2 days at 30°C, 200 rpm.
5 µl of CMdum liquid medium (without uracil) supplemented with 2% raffinose and 300 p,m of various fatty acids were then inoculated with the precultures to an OD6oo of 0.05. The expression was induced by addition of 2% (w/v) galactose. The cultures were incubated for a further 96 hours at 20°C.
Example 16: Cloning of expression plasmids for the seed-specific expression in plants
A further transformation vector based on pSUN-USP was generated for the transformation of plants. To this end, Notl cleavage sites were introduced at the 5' and 3' termini of the coding sequences, using PCR. The corresponding primer sequences are derived from the 5' and 3' regions of OtEloi and OtElo2.
Composition of the PCR mix (50 µl):
5.00 µl template cDNA
5.00 µl 10x buffer (Advantage polymerase)+ 25mM MgCI2
5.00 µl 2mM dNTP
1.25 µl of each primer (10 pmol/ µl)
0.50 µl Advantage polymerase
The Advantage polymerase from Clontech was employed. PCR reaction conditions:

Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35
The PCR products were incubated with the restriction enzyme Notl for 16 hours at 37°C. The plant expression vector pSUN300-USP was incubated in the same manner. Thereafter, the PCR products and the vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit following the manufacturer's instructions. Thereafter, vector and PCR products were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmids pSUN-OtELO1 and pSUN-OtELO2 were verified by sequencing.
pSUN300 is a derivative of plasmid pPZP (Hajdukiewicz,P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989-994). pSUN-USP originated from pSUN300, by inserting a USP promoter into pSUN300 in the form of an EcoRI fragment. The polyadenylation signal is that of the Ostreococcys gene from the A. tumefaciens Ti plasmid (ocs-Terminator, Genbank Accession V00088) (De Greve,H., Dhaese,P., Seurinck,J., Lemmers,M., Van Montagu,M. and Schell,J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octoµl ne synthase gene J. Mol. Appl. Genet. 1 (6), 499-511 (1982)). The USP promoter corresponds to nucleotides 1 to 684 (Genbank Accession X56240), where part of the noncoding region of the USP gene is present in the promoter. The promoter fragment which is 684 base pairs in size was amplified by a PCR reaction and standard methods with the aid of a synthesized primer and by means of a commercially available T7 standard primer (Stratagene). (Primer sequence: 5'-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-31, SEQ ID NO: 164).
The PCR fragment was recut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator. This gave rise to the plasmid with the name pSUN-USP. The construct was used for the transformation of Arabidopsis thaliana, oilseed rape, tobacco and linseed.
Example 17: Expression of OtELOl and OtELO2 in yeasts

Yeasts which had been transformed with the plasmids pYES3, pYES3-OtELO1 and pYES3-OtELO2 as described in Example 15 were analyzed as follows:
The yeast cells from the main cultures were harvested by centrifugation (100 x g, 5 min, 20°C) and washed with 100 mM NaHCO3) pH 8.0 to remove residual medium and fatty acids. Starting with the yeast cell sediments, fatty acid methyl esters (FAMEs) were prepared by acid methanolysis. To this end, the cell sediments were incubated for one hour at 80°C together with 2 µl of 1 N methanolic sulfuric acid and 2% (v/v) of dimethoxypropane. The FAMEs were extracted twice with petroleum ether (PE). To remove nonderivatized fatty acids, the organic phases were washed in each case once with 2 µl of 100 mM NaHCO3) pH 8.0 and 2 µl of distilled water. Thereafter, the PE phases were dried with Na2SO4, evaporated under argon and taken up in 100 \JL\ of PE. The samples were separated on a DB-23 caµl llary column (30 m, 0.25 mm, 0.25 pm, Agilent) in a Hewlett-Packard 6850 gas chromatograph equipped with flame ionization detector. The conditions for the GLC analysis were as follows: the oven temperature was programmed from 50°C to 250°C with an increment of 5°C/min and finally 10 min at 250°C (holding).
The signals were identified by comparing the retention times with corresponding fatty acid standards (Sigma). The methodology is described for example in Naµl er and Michaelson, 2001, Liµl ds. 36(8):761-766; Sayanova et al., 2001, Journal of Experimental Botany. 52(360): 1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218.
Example 18: Functional characterization of OtELOl and OtELO2:
The substrate specificity of OtELol could be determined after expression and the feeding of different fatty acids (Tab. 8). The substrates fed can be detected in large amounts in all of the transgenic yeasts. The transgenic yeasts demonstrated the synthesis of novel fatty adds, the products of the OtEloi reaction. This means that the gene OtEloi has been expressed functionally.
It can be seen from Table 7 that OtEloi has a narrow substrate specificity. OtEloi was only capable of elongating the C20-fatty acids eicosapentaenoic acid (Figure 7) and arachidonic acid (Figure 8), but preferred the ω3-desaturated eicosapentaenoic acid.

Table 8 shows the substrate specificity of the elongase OtEloi for C20-polyunsaturated fatty acids with one double bond in ∆5-position in comparison with various fatty acids.
The yeasts which had been transformed with the vector pOTE1 were cultured in minimal medium in the presence of the fatty acids detailed. The fatty acid methyl esters were synthesized by subjecting intact cells to acid methanolysis. Thereafter, the FAMEs were analyzed via GLC. Each value represents the mean (n=3) ± standard deviation.
The substrate specificity of OtELo2 (SEQ ID NO: 81) could be determined after expression and the feeding of different fatty acids (Tab. 9). The substrates fed could be detected in large amounts in all of the transgenic yeasts. The transgenic yeasts demonstrated the synthesis of novel fatty acids, the products of the OtElo2 reaction. This means that the gene OtElo2 has been expressed functionally.

Table 9 shows the substrate specificity of the eiongase OtEIo2 for various fatty acids.
The yeasts which had been transformed with the vector pOTE2 were cultured in minimal medium in the presence of the fatty acids detailed. The fatty acid methyl esters were synthesized by subjecting intact cells to acid methanolysis. Thereafter, the FAMEs were analyzed via GLC. Each value represents the mean (n=3) ± standard deviation.
The enzymatic activity shown in Table 9 clearly demonstrates that OTELO2 is a ∆6-elongase.
Example 19: Cloning genes from Thalassiosira pseudonana
The search for conserved regions in the protein sequences with the aid of the eiongase genes with ∆5-elongase activity or ∆6-elongase activity which are shown in the application allowed the identification of two sequences with suitable motifs in a Thalassiosira pseudonana sequence database (genomic sequences). The sequences were the following:

A 2 I culture of T. pseudonana was grown in f/2 medium (Guillard, R.R.L 1975. Culture of phytoplankton for feeding marine invertebrates. In Culture of Marine Invertebrate Animals (Eds. Smith, W.L. and Chanley, M.H.), Plenum Press, New York, pp 29-60) for 14 d (= days) at a light intensity of 80 E/cm2. After the cells had been spun down, RNA was isolated with the aid of the RNAeasy Kit from Quiagen (Valencia, CA, US) following the manufacturer's instructions. The mRNA was subjected to reverse transcription using the Marathon cDNA Amplification Kit (BD Biosciences) and adaptors were ligated in accordance with the manufacturer's instructions. Then, the cDNA library was used for the PCR for cloning expression plasmids by means of 5'- and 3'-RACE (raµl d amplification of cDNA ends).
Example 20: Cloning expression plasmids for the heterologous expression in yeasts
The relevant primer pairs were selected in such a way that they bore the yeast consensus sequence for highly efficient translation (Kozak, Cell 1986, 44:283-292) next to the start codon. The amplification of the TpElo DNAs was carried out in each case using 1 jil of cDNA, 200 ^m of dNTPs, 2.5 U of Advantage polymerase and 100 pmol of each primer in a total volume of 50 yd. The PCR conditions were as follows: first denaturation for 5 minutes at 95°C, followed by 30 cycles of 30 seconds at 94°C, 1 minute at 55°C and 2 minutes at 72°C, and a last elongation step of 10 minutes at 72°C.
To clone the sequence for the heterologous expression in yeasts, the following oligonucleotides were used for the PCR reaction:

*F=forward primer, R=reverse primer
The PCR products were incubated for 30 minutes at 21 °C with the yeast expression vector pYES2.1-TOPO (Invitrogen) following the manufacturer's instructions. The PCR product is ligated into the vector by means of a T overhang and activity of a topoisomerase (Invitrogen). After incubation, E. coli DH5a cells were transformed. Suitable clones were identified by PCR, the plasmid DNA was isolated by means of Qiagen DNAeasy Kit and verified by sequencing. The correct sequence was then transformed into the Saccharomyces strain INVSd (Invitrogen) by electroporation (1500 V). As a control, the blank vector pYES2.1 was transformed in parallel. The yeasts were subsequently plated onto complete uracil dropout minimal medium supplemented with 2% glucose. Cells which were capable of growing in the medium without uracil thus comprise the corresponding plasmids pYES2.1, pYES2.1-TpELO1, pYES2.1-TpELO2 and pYES2.1-TpELO3. After the selection, in each case two transformants were selected for further functional expression.
Example 21: Cloning expression plasmids for the seed specific expression in plants
A further transformation vector based on pSUN-USP is generated for the transformation of plants. To this end, Notl cleavage sites are introduced at the 5' and 3' termini of the coding sequences, using the following primer pair:
PSUN-TPELO1
Forward: 5'-GCGGCCGCACCATGTGCTCACCACCGCCGTC (SEQ ID NO: 152)
Reverse: 3'-GCGGCCGCCTACATGGCACCAGTAAC (SEQ ID NO: 153)
PSUN-TPELO2
Forward: 5'-GCGGCCGCACCATGTGCTCATCACCGCCGTC (SEQ ID NO: 154)
Reverse: 3'-GCGGCCGCCTACATGGCACCAGTAAC (SEQ ID NO: 155)

corresponds to nucleotides 1 to 684 (Genbank Accession X56240), where part of the noncoding region of the USP gene is present in the promoter. The promoter fragment which is 684 base pairs in size was amplified by a PCR reaction and standard methods with the aid of a synthesized primer and by means of a commercially available T7 standard primer (Stratagene).
(Primer sequence:
5-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-3'); SEQ ID NO: 151).
The PCR fragment was recut with EcoRl/Sall and inserted into the vector pSUN300 with OCS terminator. This gave rise to the plasmid with the name pSUN-USP. The construct was used for the transformation of Arabidopsis thaliana, oilseed rape, tobacco and linseed.
Liµl ds were extracted from yeasts and seeds as described for Example 6. Example 22: Expression of TpELOl, TpELO2 and TpELO3 in yeasts
Yeasts which had been transformed with the plasmids pYES2, pYES2-TpELO1, pYES2-TpELO2 and pYES2-TpELO3 as in Example 4 were analyzed as follows:
The yeast cells from the main cultures were harvested by centrifugation (100 x g, 5 min, 20°C) and washed with 100 mM NaHCO3, pH 8.0 in order to remove residual medium and fatty acids. Fatty acid methyl esters (FAMEs) were prepared from the yeast cell sediments by acid methanolysis. To this end, the cell sediments were incubated for 1 hour at 80°C with 2 µl of 1 N methanolic sulfuric acid and 2% (v/v) dimethoxypropane. The FAMEs were extracted by twice extracting with petroleum ether (PE). To remove nonderivatized fatty acids, the organic phases were washed in each case once with 2 µl of 100 mM NaHCO3l pH 8.0, and 2 µl of distilled water. Thereafter, the PE phases were dried with Na2SO4, evaporated under argon and taken up in 100 µl of PE. The samples were separated on a DB-23 caµl llary column (30 m, 0.25 mm, 0.25 jxm, Agilent) in a Hewlett-Packard 6850 gas chromatograph with flame ionization detector. The conditions for the GLC analysis were as follows: the oven temperature was programmed from 50°C to 250°C with an increment of 5°C/min and finally 10 minutes at 250°C (holding).
The signals were identified by comparing the retention times with corresponding fatty

acid standards (Sigma). The methodology is described for example in Naµl er and Michaelson, 2001, Liµl ds 36(8):761-766; Sayanova et al.f 2001, Journal of Experimental Botany, 52(360):1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218.
Example 23: Functional characterization of TpELOl and TpELO3:
The substrate specificity of TpELOl could be determined after expression and the feeding of different fatty acids (Figure 9). The substrates fed can be detected in large amounts in all of the transgenic yeasts. The transgenic yeasts demonstrated the synthesis of novel fatty acids, the products of the TpEloi reaction. This means that the geneTpEloi has been expressed functionally.
It can be seen from Table 10 that TpEloi shows a narrow substrate specificity. TpEloi was only capable of elongating the C20-fatty acids eicosapentaenoic acid and arachidonic acid, but preferred the ω3-desaturated eicosapentaenoic acid.
The yeasts which had been transformed with the vector pYES2-TpELO1 were cultured in minimal medium in the presence of the fatty acids detailed. The fatty acid methyl ) esters were synthesized by subjecting intact cells to acid methanolysis. Thereafter, the FAMEs were analyzed via GLC.
Table 10: Expression of TpELOl in yeast Columns 1 and 3 show the control reactions for columns 2 (fed: 250 \im 20:4 ∆5,8,11,14) and 4 (fed: 250 nm 20:4 ∆5,8,11,14,17).

The substrate specificity of TpElo3 could be determined after expression and the feeding of different fatty acids (Figure 10). The substrates fed can be detected in large amounts in all of the transgenic yeasts. The transgenic yeasts demonstrated the synthesis of novel fatty acids, the products of the TpElo3 reaction. This means that the gene TpElo3 has been expressed functionally.
It can be seen from Table 11 that TpElo3 shows a narrow substrate specificity. TpElo3 was only capable of elongating the C18-fatty acid y-linolenic acid and stearidonic acid, but preferred the ω3-desaturated stearidonic acid.
The yeasts which had been transformed with the vector pYES2-TpELO3 were cultured in minimal medium in the presence of the fatty acids detailed. The fatty acid methyl esters were synthesized by subjecting intact cells to acid methanolysis. Thereafter, the FAMEs were analyzed via GLC.
Table 11: Expression of TpELO3 in yeast. Column 1 shows the fatty acid profile of yeast without feeding. Column 2 shows the control reaction. In columns 3 to 6, the following were fed: y-Iinolenic acid, stearidonic acid, arachidonic acid and eicosapentaenoic acid (250 ^m of each fatty acid).

Example 24: Cloning and expression plasmid for the heterologous expression of the µl -omega3Des in yeasts
For the heterologous expression in yeasts, the µl -omega3Des clone was cloned into the yeast expression vector pYES3 via PCR, using suitable µl -omega3Des-specific primers. Here, exclusively the open reading frame, of the gene, which encodes the µl -omega3Des protein was amplified and provided with two cleavage sites for cloning into the pYES3 expression vector:
Forward Primer: 5'-TAAGCTTACATGGCGACGAAGGAGG (SEQ ID NO: 149) Reverse Primer 5'-TGGATCCACTTACGTGGACTTGGT (SEQ ID NO: 150)
Composition of the PCR mix (50 µl):
5.00 µl template cDNA
5.00 µl 10x buffer (Advantage polymerase)+ 25mM MgCI2
5.00 µl 2mM dNTP
1.25 µl of each primer (10 pmol/pl of the 5'ATG primer and the 3' Stopp primer)
0.50 µl Advantage polymerase
The Advantage polymerase from Clontech was employed. PCR reaction conditions:
Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35
The PCR product was incubated with the restriction enzymes Hindlll and BamHI for 2 hours at 37°C. The yeast expression vector pYES3 (Invitrogen) was incubated in the same manner. Thereafter, the 1104 bp PCR product and the vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit following the manufacturer's instructions. Thereafter, vector and desaturase cDNA were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmid pYES3-µl -omega3Des was verified by sequencing and transformed into the Saccharomyces strain INVSd (Invitrogen) by means of electroporation (1500 V). pYES3 was transformed in parallel to act as a control. Thereafter, the yeasts were plated onto complete minimal dropout tryptophan medium supplemented with 2%

glucose. Cells which were capable of growing in the medium without tryptophan thus comprise the relevant plasmids pYES3, pYES3-µl -omega3Des. Following selection, in each case two transformants were selected for the further functional expression.
Example 25: Cloning expression plasmids for the seed specific expression in plants
A further transformation vector based on pSUN-USP was generated for the transformation of plants. To this end, Notl cleavage sites were introduced at the 5' and 31 termini of the coding sequence, using the following primer pair
PSUN-µl -omega3Des
Reverse: S'-GCGGCCGCTTACGTGGACTTGGTC (SEQ ID NO: 147)
Forward: 5'-GCGGCCGCatGGCGACGAAGGAGG (SEQ ID NO: 148)
Composition of the PCR mix (50 µl):
5.00 µl template cDNA
5.00 µl 10x buffer (Advantage polymerase)+ 25mM MgCI2
5.00 µl 2mM dNTP
1.25 µl of each primer (10 pmol/µl)
0.50 µl Advantage polymerase
The Advantage polymerase from Clontech was employed. PCR reaction conditions:
Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35
The PCR products were incubated with the restriction enzyme Notl for 4 hours at 37°C. The plant expression vector pSUN300-USP was incubated in the same manner. Thereafter, the PCR products and the 7624 bp vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit following the manufacturer's instructions. Thereafter, vector and PCR products were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmid pSUN-µl omega3Des was verified by sequencing.

Example 26: Expression of µl -omega3Des in yeasts
Yeasts which had been transformed with the plasmid pYES3 or pYES3-µl -omega3Des, as described in Example 24, were analyzed as follows:
The yeast cells from the main cultures were harvested by centrifugation (100 x g, 5 min, 20°C) and washed with 100 mM NaHCO3, pH 8.0 in order to remove residual medium and fatty acids. Fatty acid methyl esters (FAMEs) were prepared from the yeast cell sediments by acid methanolysis. To this end, the cell sediments were incubated for 1 hour at 80°C with 2 µl of 1 N methanolic sulfuric acid and 2% (v/v) dimethoxypropane. The FAMEs were extracted by twice extracting with petroleum ether (PE). To remove nonderivatized fatty acids, the organic phases were washed in each case once with 2 µl of 100 mM NaHCO3, pH 8.0, and 2 µl of distilled water. Thereafter, the PE phases were dried with Na2SO4, evaporated under argon and taken up in 100 (j.l of PE. The samples were separated on a DB-23 caµl llary column (30 m, 0.25 mm, 0.25 p,m, Agilent) in a Hewlett-Packard 6850 gas chromatograph with flame ionization detector. The conditions for the GLC analysis were as follows: the oven temperature was programmed from 50°C to 250°C with an increment of 5°C/min and finally 10 minutes at 250°C (holding).
The signals were identified by comparing the retention times with corresponding fatty acid standards (Sigma). The methodology is described for example in Naµl er and Michaelson, 2001, Liµl ds 36(8):761-766; Sayanova et al., 2001, Journal of Experimental Botany, 52(360): 1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et at., 1998, FEBS Letters. 439(3):215-218.
Example 23: Functional characterization of µl -omega3Des:
The substrate specificity of µl -omega3Des could be determined after expression and the feeding of different fatty acids (Figures 12 to 18). The substrates fed are present in large amounts in all of the transgenic yeasts, which proves that these fatty acids have been taken up into the yeasts. The transgenic yeasts demonstrate the synthesis of novel fatty acids, the products of the µl -omega3Des reaction. This means that the gene µl -omega3Des has been expressed functionally.
Figure 12 represents the desaturation of linoleic acid (18:2 w6-fatty acid) to give a-linolenic acid (18:3 ω3-fatty acid) by µl -omega3Des. The fatty acid methyl esters were synthesized by subjecting intact cells which had been transformed with the blank vector pYES2 (Figure 12 A) or the vector pYES3- µl -omega3Des (Figure 12 B) to acid methanolysis. The yeasts were cultured in minimal medium in the presence of 18:2∆9>12-

fatty acid (300 jim). Thereafter, the FAMEs were analyzed via GLC.
Figure 13 represents the desaturation of y-Iinolenic acid (18:3 ω6--fatty acid) to give stearidonic acid (18:4 ω3-fatty acid) by µl -omega3Des. The fatty acid methyl esters were synthesized by subjecting intact cells which had been transformed with the blank vector pYES2 (Figure 13 A) or the vector pYes3-µl -omega3Des (Figure 13 B) to acid methanoiysis. The yeasts were cultured in minimal medium in the presence of y-C18:3∆6i9i12-fatty acid (300 jim). Thereafter, the FAMEs were analyzed via GLC.
Figure 14 represents the desaturation of C20:2-ω6--fatty acid to give C20:3-ω3-fatty acid by µl -omega3Des. The fatty acid methyl esters were synthesized by subjecting, intact cells which had been transformed with the blank vector pYES2 (Figure 14 A) or the vector pYes3-µl -omega3Des (Figure 14 B) to acid methanoiysis. The yeasts were cultured in miminal medium in the presence of C20:2A11'14-fatty acid (300 p.m). Thereafter, the FAMEs were analyzed via GLC.
Figure 15 represents the desaturation of C20:3-ω6--fatty acid to give C20:4-oo3-fatty acid by µl -omega3Des. The fatty acid methyl esters were synthesized by subjecting intact cells which had been transformed with the blank vector pYES2 (Figure 15 A) or the vector pYes3-µl -omega3Des (Figure 15 B) to acid methanoiysis. The yeasts were cultured in miminal medium in the presence of C20:3A8'11i14-fatty acid (300 jam). Thereafter, the FAMEs were analyzed via GLC.
Figure 16 shows the desaturation of arachidonic acid (C20:4-ω6--fatty acid) to give eicosapentaenoic acid (C20:5-cj3-fatty acid) by µl -omega3Des.
The fatty acid methyl esters were synthesized by subjecting intact cells which had been transformed with the blank vector pYES2 (Figure 16 A) or the vector pYes3-µl -omega3Des (Figure 16 B) to acid methanoiysis. The yeasts were cultured in minimal medium in the presence of C20:4∆5i8'11t14-fatty acid (300 urn). Thereafter, the FAMEs were analyzed via GLC.
Figure 17 represents the desaturation of docosatetraenoic acid (C22:4-w6-fatty acid) to give docosapentaenoic acid (C22:5-ω3-fatty acid) by µl -omega3Des. The fatty acid methyl esters were synthesized by subjecting intact cells which had been transformed with the blank vector pYES2 (Figure 17 A) or the vector pYes3-µl -omega3Des (Figure 17 B) to add methanoiysis. The yeasts were cultured in minimal medium in the presence of C22:4∆7l1Of13l16-fatty acid (300 jim). Thereafter, the FAMEs were analyzed

via GLC.
The substrate specificity of µl -omega3Des with regard to different fatty acids can be seen from Figure 18. The yeasts which had been transformed with the vector pYes3-µl -omega3Des were cultured in minimal medium in the presence of the fatty acids detailed. The fatty acid methyl esters were synthesized by subjecting intact cells to acid methanolysis. Thereafter, the FAMEs were analyzed via GLC. Each value represents a mean of three measurements. The conversion rates (% desaturation) were calculated using the formula: [product]/[product]+[substrate]*100.
As described in Example 9, µl -omega3Des can also be used for generating transgenic plants. Then, the liµl ds can be extracted from the seeds of these plants as described under Example 6.
Example 28: Cloning desaturase genes from Ostreococcus tauri
The search for conserved regions in the protein sequences with the aid of conserved motifs (His boxes, Domergue et al. 2002, Eur. J. Biochem. 269, 4105-4113) allowed the identification of five sequences with corresponding motifs in an Ostreococcus tauri sequence database (genomic sequences). The sequences were the following:

The alignments for finding homologies of the individual genes were carried out using the tBLASTn algorithm (Altschul et al., J. Mol. Biol. 1990, 215: 403-410).
The cloning procedure was as follows:
40 µl of an Ostreococcus tauri culture in the stationary phase were spun down, resuspended in 100 \i\ of double-distilled water and stored at -20°C. The respective genomic DNAs were amplified on the basis of the PCR process. The relevant primer

pairs were selected in such a way that they bore the yeast consensus sequence for highly efficient translation (Kozak, Cell 1986, 44:283-292) next to the start codon. The amplification of the OtDes DNAs was carried out in each case using 1 nl of defrosted cells, 200 urn of dNTPs, 2.5 U Taq polymerase and 100 pmol of each primer in a total volume of 50 JJ.I. The PCR conditions were as follows: first denaturation for 5 minutes at 95°C, followed by 30 cycles of 30 seconds at 94°C, 1 minute at 55°C and 2 minutes at 72°C, and a last elongation step of 10 minutes at 72°C.
The following primers were employed in the PCR:
OtDes6.1 Forward: 5'ggtaccacataatgtgcgtggagacggaaaataacg3' (SEQ ID NO: 145) OtDes6.1 Reverse: 5'ctcgagttacgccgtctttccggagtgttggcc3' (SEQ ID NO: 146)
Example 29: Cloning expression plasmids for the heterologous expression in yeasts:
To characterize the function of the desaturase OtDes6.1 (=∆6-desaturase) from Ostreococcus tauri, the open reading frame of the DNA was cloned downstream of the galactose-inducible GAL1 promoter of pYES2.1/V5-His-TOPO (Invitrogen), giving rise to the corresponding clone pYES2.1-OtDes6.1. Further desaturase genes from Ostreococcus can be cloned analogously.
The Saccharomyces cerevisiae strain 334 was transformed by electroporation (1500 v) with the vector pYES2.1-OtDes6.1. A yeast which was transformed with the blank vector pYES2 was used as the control. The transformed yeasts were selected on complete minimal dropout uracil medium (CMdum) agar plates supplemented with 2% glucose. After the selection, in each case three transformants were selected for the further functional expression.
To express the OtDes6.1 desaturase, precultures of in each case 5 µl of dropout uracil CMdum liquid medium supplemented with 2% (w/v) raffinose were inoculated with the selected transformants and incubated for 2 days at 30'C, 200 rpm. 5 µl of CMdum liquid medium (without uracil) supplemented with 2% raffinose and 300 ,m of vanous fatty acids were then inoculated with the precultures to an OD600 of 0.05. Express.on was induced by addition of 2% (w/v) galactose. The cultures were incubated for a further 96 hours at 20°C.
Example 30. Cloning of expression plasmids for the seed-specific expression in
plants

A further transformation vector based on pSUN-USP is generated for the transformation of plants. To this end, Notl cleavage sites are introduced at the 5' and 3' termini of the coding sequences, using PCR. The corresponding primer sequences are derived from the 5' and 3' regions of the desaturases.
Composition of the PCR mix (50 \s\):
5.00 µl template cDNA
5.00 \}\ 10x buffer (Advantage polymerase)+ 25mM MgCI2
5.00 \s\ 2mM dNTP
1.25 µl of each primer (10 pmol/µl
0.50 µl Advantage polymerase
The Advantage polymerase from Clontech was employed.
PCR reaction conditions: Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35
The PCR products were incubated with the restriction enzyme Notl for 16 hours at 37°C. The plant expression vector pSUN300-USP was incubated in the same manner. Thereafter, the PCR products and the vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit following the manufacturer's instructions. Thereafter, vector and PCR products were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmids were verified by sequencing.
pSUN300 is a derivative of plasmid pPZP (Hajdukiewicz.P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989-994). pSUN-USP originated from pSUN300, by inserting a USP promoter into pSUN300 in the form of an EcoRI fragment. The polyadenylation signal is that of the Ostreococcus gene from the A. tumefaciens Ti plasmid (ocs-Terminator, Genbank Accession V00088) (De Greve,H., Dhaese,P., Seurinck,J., Lemmers,M., Van Montagu,M. and Schell,J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octoµl ne

synthase gene J. Mol. Appl. Genet. 1 (6), 499-511 (1982)). The USP promoter corresponds to nucleotides 1 to 684 (Genbank Accession X56240), where part of the noncoding region of the USP gene is present in the promoter. The promoter fragment which is 684 base pairs in size was amplified by a PCR reaction and standard methods with the aid of a synthesized primer and by means of a commercially available T7 standard primer (Stratagene). (Primer sequence: 5'-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-3', SEQ ID NO: 144).
The PCR fragment was recut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator. This gave rise to the pfasmid with the name pSUN-USP, The construct was used for the transformation of Arabidopsis thaliana, oilseed rape, tobacco and linseed.
Example 31: Expression of OtDes6.1 in yeasts
Yeasts which had been transformed with the plasmids pYES2, pYES2- OtDes6.2 as described in Example 4 were analyzed as follows:
The yeast cells from the main cultures were harvested by centrifugation (100 x g, 5 min, 20°C) and washed with 100 mM NaHCO3, pH 8.0 to remove residual medium and fatty acids. Starting with the yeast cell sediments, fatty acid methyl esters (FAMEs) were prepared by acid methanolysis. To this end, the cell sediments were incubated for one hour at 80°C together with 2 µl of 1 N methanolic sulfuric acid and 2% (v/v) of dimethoxypropane. The FAMEs were extracted twice with petroleum ether (PE). To remove nonderivatized fatty acids, the organic phases were washed in each case once with 2 µl of 100 mM NaHCO3, pH 8.0 and 2 µl of distilled water. Thereafter, the PE phases were dried with Na2SO4j evaporated under argon and taken up in 100 µl of PE. The samples were separated on a DB-23 caµl llary column (30 m, 0.25 mm, 0.25 jjm, Agilent) in a Hewlett-Packard 6850 gas chromatograph equipped with flame ionization detector. The conditions for the GLC analysis were as follows: the oven temperature was programmed from 50°C to 250°C with an increment of 5°C/min and finally 10 min at 250°C (holding).
The signals were identified by comparing the retention times with corresponding fatty acid standards (Sigma). The methodology is described for example in Naµl er and Michaelson, 2001, Liµl ds. 36(8):761-766; Sayanova et al., 2001, Journal of Experimental Botany. 52(360): 1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218.

Example 32: Functional characterization of desaturases from Ostreococcus:
The substrate specificity of desaturases can be determined after expression in yeast (see examples Cloning desaturase genes, Yeast expression) by feeding by means of different yeasts. Descriptions for determining the individual activities are found in WO 93/11245 for A15-desaturases, WO 94/11516 for ∆12-desaturases, WO 93/06712, US 5,614,393, US 5614393, WO 96/21022, WO 0021557 and WO 99/27111 for ∆6-desaturases, Qiu et al. 2001, J. Biol. Chem. 276, 31561-31566 for ∆4-desaturases, Hong et al. 2002, Liµl ds 37, 863-868 for ∆5-desaturases.
Table 12 represents the substrate specificity of the desaturase OtDes6.1 with regard to different fatty acids. The substrate specificity of OtDes6.1 was determined after expression and feeding of various fatty acids. The substrates which have been fed can be detected in large amounts in all of the transgenic yeasts. The transgenic yeasts demonstrated the synthesis of novel fatty acids, the products of the OtDes6.2 reaction (Fig. 20). This means that the gene OtDes6.1 has been expressed functionally.
The yeasts which had been transformed with the vector pYES2-OtDes6.1 were cultured in minimal medium in the presence of the fatty acids detailed. The fatty acid methyl esters were synthesized by subjecting intact cells to acid methanolysis. Thereafter, the FAMEs were analyzed via GLC. Each value represents the mean (n=3) ± standard deviation. The activity corresponds to the conversion rate calculated using the formula [substrate/(substrate+product)*100].
It can be seen from Table 12 that OtDes6.1 shows substrate specificity for linoleic and linolenic acid (18:2 and 18:3) since the highest activities are obtained with these fatty acids. In contrast, the activity for oleic acid (18:1) and pal.,itoleic acid (16:1) is markedly lower. The preferred conversion of iinoleic and linolenic acid demonstrates that this desaturase is suitable for the production of polyunsaturated fatty acids.

Figure 20 shows the conversion of linoleic acid by OtDes6.1. The FAMEs were analyzed via gas chromatography. The substrate which has been fed (C18:2) is converted into y-C18:3. Both the starting material and the resulting product are indicated by arrows.
Figure 21 represents the conversion of linoleic acid (= LA) and a-Iinolenic acid (= ALA) in the presence of OtDes6.1 to give y-linolenic acid (= GLA) and stearidonic acid (= STA), respectively (Figure 21 A and C). Moreover, Figure 21 shows the conversion of linoleic acid (= LA) and a-linolenic acid (= ALA) in the presence of the ∆6-desaturase OtDes6.1 together with the Physcomitrella patens ∆6-elongase PSE1 (Zank et al. 2002, Plant J. 31:255-268) and the Phaeodactylum tricornutum ∆5-desaturase PtD5 (Domergue et al. 2002, Eur. J. Biochem. 269, 4105-4113) to give dihomo-y-linoienic acid (= DHGLA) and arachidonic acid (= ARA, Figure 21 B) and dihomostearidonic acid (= DHSTA) and eicosapentaenoic acid (= EPA, Figure 21 D), respectively. Figure 21 shows clearly that the reaction products GLA and STA of the ∆6-desaturase OtDes6.1 in the presence of the ∆6-elongase PSE1 is elongated virtually quantitatively to give DHGLA and DHSTA, respectively. The subsequent desaturation by the ∆5-desaturase PtD5 to give ARA and EPA, respectively, also proceeds smoothly. Approximately 25-30% of the elongase product is desaturated (Figure 21 B and D).
Table 13 which follows gives an overview of the Ostreococcus desaturases which have been cloned:

Example 33: Cloning desaturase genes from Thalassiosira pseudonana
The search for conserved regions in the protein sequences with the aid of conserved motifs (His boxes, see motifs) allowed the identification of six sequences with corresponding motifs in an Thalassiosira pseudonana sequence database (genomic sequences). The sequences were the following:

The cloning procedure was as follows:
40 µl of an Thalassiosira pseudonana culture in the stationary phase were spun down, resuspended in 100 \i\ of double-distilied water and stored at -20°C. The respective genomic DNAs were amplified on the basis of the PCR method. The relevant primer pairs were selected in such a way that they bore the yeast consensus sequence for highly efficient translation (Kozak, Cell 1986, 44:283-292) next to the start codon. The amplification of the TpDes DNAs was carried out in each case using 1 jal of defrosted cells, 200 µm of dNTPs, 2.5 U Taq polymerase and 100 pmol of each primer in a total volume of 50 µl The PCR conditions were as follows: first denaturation for 5 minutes at 95°C, followed by 30 cycles of 30 seconds at 94°C, 1 minute at 55°C and 2 minutes at 72°C, and a last elongation step of 10 minutes at 72°C.
Example 34: Cloning expression plasmids for the heterologous expression in yeasts:
To characterize the function of the desaturases from Thalassiosira pseudonana, the open reading frame of the respective DNA was cloned downstream of the galactose-inducible GAL1 promoter of pYES2. W5-His-TOPO (Invitrogen), giving rise to the corresponding pYES2.1 clone.
The SaccharQmyces cerevisiae strain 334 is transformed by electroporation (1500 v)

with the vectors pYES2.1-TpDesaturasen. A yeast which is transformed with the blank vector pYES2 is used as the control. The transformed yeasts are selected on complete minimal dropout uracil medium (CMdum) agar plates supplemented with 2% glucose. After the selection, in each case three transformants are selected for the further functional expression.
To express the Tp desaturases, initially precultures of in each case 5 µl of dropout uracil CMdum liquid medium supplemented with 2% (w/v) raffinose are inoculated with the selected transformants and incubated for 2 days at 30°C, 200 rpm. 5 µl of liquid CMdum medium (without uracil) supplemented with 2% raffinose and 300 jam of various fatty acids are then inoculated with the precultures to an OD6oo of 0.05. The expression is induced by addition of 2% (w/v) galactose. The cultures are incubated for a further 96 hours at 20°C.
Example 35: Cloning of expression plasmids for the seed-specific expression in
plants
A further transformation vector based on pSUN-USP is generated for the transformation of plants. To this end, Notl cleavage sites are introduced at the 5' and 3' termini of the coding sequences, using PCR. The corresponding primer sequences are derived from the 5' and 3' regions of the desaturases.
Composition of the PCR mix (50 yl):
5.00 µl template cDNA
5.00 fjl 10x buffer (Advantage polymerase)+ 25mM MgCI2
5.00 µl 2mM dNTP
1.25 µl of each primer (10 pmol/µl )
0.50 µl Advantage polymerase
The Advantage polymerase from Clontech was employed.
PCR reaction conditions: Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35

The PCR products are incubated with the restriction enzyme Notl for 16 hours at 37°C. The plant expression vector pSUN300-USP is incubated in the same manner. Thereafter, the PCR products and the vector are separated by agarose gel electrophoresis and the corresponding DNA fragments are excised. The DNA was purified by means of the Qiagen Gel Purification Kit following the manufacturer's instructions. Thereafter, vector and PCR products are ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmids are verified by sequencing.
pSUN300 is a derivative of plasmid pPZP (Hajdukiewicz,P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989-994). pSUN-USP originated from pSUN300, by inserting a USP promoter into pSUN300 in the form of an EcoRI fragment. The polyadenylation signal is the OCS gene from the A. tumefaciens Ti plasmid (ocs-Terminator, Genbank Accession V00088) (De Greve.K, Dhaese.P., Seurinck,J., Lemmers,M., Van Montagu,M. and Schell,J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octoµl ne synthase gene J. Mol. Appl. Genet. 1 (6), 499-511 (1982)). The USP promoter corresponds to nucleotides 1 to 684 (Genbank Accession X56240), where part of the noncoding region of the USP gene is present in the promoter. The promoter fragment which is 684 base pairs in size was amplified by a PCR reaction and standard methods with the aid of a synthesized primer and by means of a commercially available T7 standard primer (Stratagene). (Primer sequence: 51-
GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-3', SEQ ID NO: 143).
The PCR fragment was recut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator. This gave rise to the plasmid with the name pSUN-USP. The construct was used for the transformation of Arabidopsis thaliana, oilseed rape, tobacco and linseed.
Example 36: Expression of Tp desaturases in yeasts
Yeasts which have been transformed with the plasmids pYES2 and pYES2-TpDesaturasen as described in Example 4 were analyzed as follows:
The yeast cells from the main cultures are harvested by centrifugation (100 x g, 5 min, 20°C) and washed with 100 mM NaHCO3, pH 8.0 to remove residual medium and fatty acids. Starting with the yeast cell sediments, fatty acid methyl esters (FAMEs) are

prepared by acid methanolysis. To this end, the cell sediments are incubated for one hour at 80°C together with 2 µl of 1 N methanolic sulfuric acid and 2% (v/v) of dimethoxypropane. The FAMEs were extracted twice with petroleum ether (PE). To remove nonderivatized fatty acids, the organic phases are washed in each case once with 2 µl of 100 mM NaHCO3, pH 8.0 and 2 µl of distilled water. Thereafter, the PE phases are dried with Na2SO4, evaporated under argon and taken up in 100 [s\ of PE. The samples are separated on a DB-23 caµl llary column (30 m, 0.25 mm, 0.25 pm, Agilent) in a Hewlett-Packard 6850 gas chromatograph equipped with flame ionization detector. The conditions for the GLC analysis are as follows: the oven temperature is programmed from 50°C to 250°C with an increment of 5°C/min and finally 10 min at 250°C (holding).
The signals are identified by comparing the retention times with corresponding fatty acid standards (Sigma). The methodology is described for example in Naµl er and Michaelson, 2001, Liµl ds. 36(8):761-766; Sayanova et al., 2001, Journal of Experimental Botany. 52(360): 1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218.
Example 37: Functional characterization of desaturases from Thalassiosira pseudonana:
The substrate specificity of desaturases can be determined after expression in yeast (see examples Cloning desaturase genes, Yeast expression) by feeding by means of different yeasts. Descriptions for determining the individual activities are found in WO 93/11245 for A15-desaturases, WO 94/11516 for ∆12-desaturases, WO 93/06712, US 5,614,393, US 5614393, WO 96/21022, WO 0021557 and WO 99/27111 for ∆6-desaturases, Qiu et al. 2001, J. Biol. Chem. 276, 31561-31566 for ∆4-desaturases, Hong et al. 2002, Liµl ds 37, 863-868 for ∆5-desaturases.
The activity of the individual desaturases is calculated from the conversion rate using the formula [substrate/(substrate+product)*100]
Tables 11 and 12 which follow give an overview of the cloned Thalassiosira pseudonana desaturases.

Table 14: Length and characteristic features of the cloned Thalassiosira pseudonana desaturases

The ∆12-desaturase genes from Ostreococcus and Thalassiosira can also be cloned analogously to the above examples.
Example 38: Cloning elongase genes from Xenopus laevis and Ciona intestinalis

The search for conserved regions (see consensus sequences, SEQ ID NO: 115 and SEQ ID NO: 116) in the protein sequences in gene databases (Genbank) with the aid of the elongase genes with ∆5-elongase activity or ∆6-elongase activity, which are detailed in the application, allowed the identification and isolation of further elongase sequences from other organisms. Further sequences were identified in each case from X. laevis and from C. intestinalis, using suitable motifs. The sequences were the following:

The cDNA clone of X. laevis was obtained from the NIH (National Institute of Health) [Genetic and genomic tools for Xenopus research: The NIH Xenopus initiative, Dev. Dyn. 225 (4), 384-391 (2002)].
The cDNA clone of C. intestinalis was obtained from the University of Kyoto [Satou, Y.t Yamada, L, Mochizuki, Y., Takatori, NM Kawashima, T., Sasaki, A., Hamagu-chi, M., Awazu, S., Yagi, K., Sasakura, Y.f Nakayama, A., Ishikawa, H., Inaba, K. and Satoh, N. "A cDNA resource from the basal chordate Ciona intestinalis" JOURNAL Genesis 33 (4), 153-154(2002)].
Example 39: Cloning expression plasmids for the heterologous expression in yeasts
The elongase DNAs were amplified in each case using 1 joJ of cDNA, 200 \xM dNTPs, 2.5 U of Advantage pplymerase and 100 pmol of each primer in a total volume of 50 ^l. The PCR conditions were as follows: first denaturation for 5 minutes at 95°C, followed by 30 cycles of 30 seconds at 94°C, 1 minute at 55°C and 2 minutes at 72°C1 and a final elongation step of 10 minutes at 72°C.
To clone the sequence for heterologous expression in yeasts, the following oligonucleotides were used for the PCR reaction:

*F=forward primer, R=reverse primer
The PCR products were incubated for 30 minutes at 21 °C with the yeast expression vector pYES2.1-TOPO (Invitrogen) following the manufacturer's instructions. The PCR product is ligated into the vector by means of a T overhang and activity of a topoisomerase (Invitrogen). After incubation, E. coli DH5a cells were transformed. Suitable clones were identified by PCR, the plasmid DNA was isolated by means of Qiagen DNAeasy Kit and verified by sequencing. The correct sequence was then transformed into the Saccharomyces strain INVSd (Invitrogen) by electroporation (1500 V). As a control, the blank vector pYES2.1 was transformed in parallel. The yeasts were subsequently plated onto complete uracil dropout minimal medium supplemented with 2% glucose. Cells which were capable of growing in the medium without uracil thus comprise the corresponding plasmids pYES2.1, pYES2.1-ELO(XI) and pYES2.1-ELO(Ci). After the selection, in each case two transformants were selected for further functional expression.
Example 40: Cloning expression plasmids for the seed-specific expression in plants
A further transformation vector based on pSUN-USP is generated for the transformation of plants. To this end, Notl cleavage sites are introduced at the 5' and 3' ends of the coding sequence, using the following primer pair:
pSUN-ELO(XI)
Forward: 5'-GCGGCCGCACCATGGCCTTCAAGGAGCTCACATC
(SEQIDNO: 125)
Reverse: 3'-GCGGCCGCCTTCAATGGTTTTTGCTTTTCAATGCACCG
(SEQIDNO: 126)
pSUN-ELO(Ci)

corresponds to nucleotides 1 to 684 (Genbank Accession X56240), where part of the noncoding region of the USP gene is present in the promoter. The promoter fragment which is 684 base pairs in size was amplified by a PCR reaction and standard methods with the aid of a synthesized primer and by means of a commercially available T7 standard primer (Stratagene).
Primer sequence:
5'-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-3' (SEQ ID NO: 129).
The PCR fragment was recut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator. This gave rise to the plasmid with the name pSUN-USP. The construct was used for the transformation of Arabidopsis thaliana, oilseed rape, tobacco and linseed.
Liµl ds were extracted from yeasts and seeds as described for Example 6. Example 41: Expression of ELO(XI) and ELO(Ci) in yeasts
Yeasts which had been transformed with the plasmids pYES2, pYES2-ELO(XI) and pYES2-ELO(Ci) as in Example 4 were analyzed as follows:
The yeast cells from the main cultures were harvested by centrifugation (100 x g, 5 min, 20°C) and washed with 100 mM NaHCO3) pH 8.0 in order to remove residual medium and fatty acids. Fatty acid methyl esters (FAMEs) were prepared from the yeast cell sediments by acid methanolysis. To this end, the cell sediments were incubated for 1 hour at 80°C with 2 µl of 1N methanolic sulfuric acid and 2% (v/v) dimethoxypropane. The FAMEs were extracted by twice extracting with petroleum ether (PE). To remove non-derivatized fatty acids, the organic phases were washed in each case once with 2 µl of 100 mM NaHCO3, pH 8.0, and 2 µl of distilled water. Thereafter, the PE phases were dried with Na2SO4, evaporated under argon and taken up in 100 p-l of PE. The samples were separated on a DB-23 caµl llary column (30 m, 0.25 mm, 0.25 jj,m, Agilent) in a Hewlett-Packard 6850 gas chromatograph with flame ionization detector. The conditions for the GLC analysis were as follows: the oven temperature was programmed from 50°C to 250°C with an increment of 5°C/min and finally 10 minutes at 250°C (holding).
The signals were identified by comparing the retention times with corresponding fatty

acid standards (Sigma). The methodology is described for example in Naµl er and Michaelson, 2001, Liµl ds 36(8):761-766; Sayanova et al., 2001, Journal of Experimental Botany, 52(360):1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et af., 1998, FEBS Letters. 439(3):215-218.
Example 42: Functional characterization of ELO(XI) and ELO(Ci):
The substrate specificity of ELO(Xl) can be determined after expression and the feeding of different fatty acids (Figure 22). The substrates fed can be detected in large amounts in all of the transgenic yeasts. The transgenic yeasts demonstrated the synthesis of novel fatty acids, the products of the ELO(XI) reaction. This means that the gene ELO(XI) has been expressed functionally.
It can be seen from Table 16 that ELO(XI) shows a broad substrate specificity. Both C18- and C20-fatty acids are elongated, but a preference for ∆5- and ∆6-desaturated fatty acids can be observed.
The yeasts which had been transformed with the vector pYES2-ELO(XI) were cultured in minimal medium in the presence of the fatty acids detailed. The fatty acid methyl esters were synthesized by subjecting intact cells to acid methanolysis. Thereafter, the FAMEs were analyzed via GLC.
Table 16: Expression of ELO(XI) in yeast. The conversion rate of different starting materials (amounts fed: in each case 250 \M) is described.

The substrate specificity of ELO(Ci) can be determined after expression and the feeding of different fatty acids (Fig. 23). The substrates fed can be detected in large amounts in all of the transgenic yeasts. The transgenic yeasts demonstrated the synthesis of novel fatty acids, the products of the ELO(Ci) reaction. This means that the gene ELO(Ci) has been expressed functionally.
Table 17: Expression of ELO(Ci) in yeast. The conversion rate of different starting materials (amounts fed: in each case 250 µm) is described.

It can be seen from Table 17 that ELO(Ci) shows a broad substrate specificity. Both C18- and C20-fatty acids are elongated, but a preference for ∆5- and ∆6-desaturated fatty acids can be observed.

The yeasts which had been transformed with the vector pYES2-ELO(Ci) were cultured in minimal medium in the presence of the fatty acids detailed. The fatty acid methyl esters were synthesized by subjecting intact cells to acid methanolysis. Thereafter, the FAMEs were analyzed via GLC.
Example 43: Cloning genes from Ostreococcus tauri
The search for conserved regions in the protein sequences with the aid of the elongase genes with ∆5-elongase activity or ∆6-elongase activity, which have been described herein, allowed the identification of in each case two sequences with corresponding motifs in an Ostreococcus tauri sequence database (genomic sequences). The sequences were the following:

OtEloi and OtElo1.2 show the highest similarity with an elongase from Danio rerio (GenBank AAN77156; approximately 26% identity), while OtElo2 and OtElo2.1 show the highest similarity with Physcomitrella Elo (PSE) [approx. 36% identity] (alignments were carried out using the tBLASTn algorithm (Altschul et al., J. Mol. Biol. 1990, 215: 403-410)).
The elongases were cloned as follows:
40 µl of an Ostreococcus tauri culture in the stationary phase were spun down, resuspended in 100 \si of double-distilled water and stored at -20°C. The respective genomic DNAs were amplified on the basis of the PCR method. The relevant primer pairs were selected in such a way that they bore the yeast consensus sequence for highly efficient translation (Kozak, Cell 1986, 44:283-292) next to the start codon. The amplification of the OtElo DNAs was carried out in each case using 1 yd of defrosted cells, 200 \xM of dNTPs, 2.5 U Taq polymerase and 100 pmol of each primer in a total volume of 50 µl . The PCR conditions were as follows: first denaturation for 5 minutes at 95°C, followed by 30 cycles of 30 seconds at 94°C, 1 minute at 55°C and 2 minutes at

72°C, and a last elongation step of 10 minutes at 72°C.
Example 44: Cloning expression plasmids for the heterologous expression in yeasts:
To characterize the function of the elongases from Ostreococcus tauri, the open reading frames of the respective DNAs were cloned downstream of the galactose-inducible GAL1 promoter of pYES2.1/V5-His-TOPO (Invitrogen), giving rise to pOTE1, pOTEI.2, pOTE2 and pOTE2.1.
The Saccharomyces cerevisiae strain 334 was transformed by electroporation (1500 V) with the vector pOTE1, pOTE1.2, pOTE2 and pOTE2.1, respectively. A yeast which was transformed with the blank vector pYES2 was used as the control. The transformed yeasts were selected on complete minimal dropout uracil medium (CMdum) agar plates supplemented with 2% glucose. After the selection, in each case three transformants were selected for the further functional expression.
To express the Ot elongases, precultures of in each case 5 µl of liquid CMdum medium supplemented with 2% (w/v) raffinose, but without uracil, were inoculated with the selected transformants and incubated for 2 days at 30°C, 200 rpm. 5 µl of liquid CMdum medium (without uracil) supplemented with 2% raffinose and 300 (im of various fatty acids were then inoculated with the precultures to an OD6oo of 0.05. The expression was induced by addition of 2% (w/v) galactose. The cultures were incubated for a further 96 hours at 20°C.
Example 45: Cloning of expression plasmids for the seed-specific expression in
plants
A further transformation vector based on pSUN-USP was generated for the transformation of plants. To this end, Notl cleavage sites were introduced at the 51 and 3' ends of the coding sequences, using PCR. The corresponding primer sequences were derived from the 5' and 3' regions of OtEloi, OtEloi .2, OtElo2 and OtElo2.1.
Composition of the PCR mix (50 µl):
5.00 µl template cDNA
5.00 µl 10x buffer (Advantage polymerase)+ 25mM MgCI2
5.00 µl 2mM dNTP
1.25 µl of each primer (10 pmol/ µl)

0.50 µl Advantage polymerase
The Advantage polymerase from Clontech was employed.
PCR reaction conditions:
Annealing temperature: 1 min 55°C
Denaturation temperature: 1 min 94°C
Elongation temperature: 2 min 72°C
Number of cycles: 35
The PCR products are incubated with the restriction enzyme Notl for 16 hours at 37°C. The plant expression vector pSUN300-USP is incubated in the same manner. Thereafter, the PCR products and the vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit following the manufacturer's instructions. Thereafter, vector and PCR products were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmids pSUN-OtELO1, pSUN-OtELO1.2, pSUN-OtELO2 and pSUN-OtELO2.2 were verified by sequencing.
pSUN300 is a derivative of plasmid pPZP (Hajdukiewicz,P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989-994). pSUN-USP originated from pSUN300, by inserting a USP promoter into pSUN300 in the form of an EcoRI fragment. The polyadenylation signal is that of the Ostreococcus gene from the A. tumefaciens Ti plasmid (ocs-Terminator, Genbank Accession V00088) (De Greve,H., Dhaese,P., Seurinck,J., Lemmers.M., Van Montagu,M. and ScheII,J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octoµl ne synthase gene J. Mol. Appl. Genet. 1 (6), 499-511 (1982)). The USP promoter corresponds to nucleotides 1 to 684 (Genbank Accession X56240), where part of the noncoding region of the USP gene is present in the promoter. The promoter fragment which is 684 base pairs in size was amplified by a PCR reaction and standard methods with the aid of a synthesized primer and by means of a commercially available T7 standard primer (Stratagene).
Primer sequence:
5-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-31, SEQ ID NO: 130.

The PCR fragment was recut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator. This gave rise to the plasmid with the name pSUN-USP. The construct was used for the transformation of Arabidopsis thaliana, oilseed rape, tobacco and linseed.
Example 46: Expression of OtEloi, OtE!o1.2, OtElo2 and OtELO2.2 in yeasts
Yeasts which had been transformed with the plasmids pYES3, pYES3»OtEIO1, pYES3-OtEIO1.2, pYES3- OtELO2 and pYES3-OtELO2.2 as described in Example 15 were analyzed as follows:
The yeast cells from the main cultures were harvested by centrifugation (100 x g, 5 min, 20°C) and washed with 100 mM NaHCO3, pH 8.0 to remove residual medium and fatty acids. Starting with the yeast cell sediments, fatty acid methyl esters (FAMEs) were prepared by acid methanolysis. To this end, the cell sediments were incubated for 1 hour at 80°C together with 2 µl of 1 N methanolic sulfuric acid and 2% (v/v) of dimethoxypropane. The FAMEs were extracted twice with petroleum ether (PE). To remove nonderivatized fatty acids, the organic phases were washed in each case once with 2 µl of 100 mM NaHCO3, pH 8.0 and 2 µl of distilled water. Thereafter, the PE phases were dried with Na2SO4, evaporated under argon and taken up in 100 µl of PE. The samples were separated on a DB-23 caµl llary column (30 m, 0.25 mm, 0.25 pm, Agilent) in a Hewlett-Packard 6850 gas chromatograph equipped with flame ionization detector. The conditions for the GLC analysis were as follows: the oven temperature was programmed from 50°C to 250°C with an increment of 5°C/min and finally 10 min at 250°C (holding).
The signals were identified by comparing the retention times with corresponding fatty acid standards (Sigma). The methodology is described for example in Naµl er and Michaelson, 2001, Liµl ds. 36(8):761-766; Sayanova et al., 2001, Journal of Experimental Botany. 52(360): 1581-1585, Sperling et al., 2001, Arch, Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218.
Example 47: Functional characterization of OtEloi, OtElol.2, OtElo2 and OtElo2.1:
The substrate specificity of OtEloi was determined after expression and feeding of different fatty acids (Table 18). The substrates which have been fed can be detected in large amounts in all transgenic yeasts. The transgenic yeasts showed the synthesis of novel fatty acids, the products of the OtEloi reaction. This means that the gene OtEloi

Table 19 shows the substrate specificity of the elongase OtElo2 and OtElo2.1 with regard to various fatty acids. OtElo2.1 shows a markedly higher activity.
The yeasts which had been transformed with the vector pOTE2 or pOTE2.1 were cultured in minimal medium in the presence of the fatty acids stated. The fatty acid methyl esters were synthesized by subjecting intact cells to acid methanolysis. Thereafter, the FAMEs were analyzed via GLC.
The enzymatic activity shown in Table 19 clearly demonstrates that OtElo2 and OtElo2.1, respectively, are a ∆6-elongase.

Figure 24 A - D shows the elongation of eicosapentaenoic acid by OtElol (B) and OtElo1.2 (D), respectively. The controls (A, C) do not show the elongation product (22:5co3).
Figure 25 A - D shows the elongation of arachidonic acid by OtElol (B) and OtElol .2 (D), respectively. The controls (A, C) do not show the elongation product (22:4oo6).
Example 48: Cloning elongase genes from Euglena gracilis and Arabidopsis thaliana
The search for conserved regions in the protein sequences with the aid of the elongase genes with ∆5-elongase activity or ∆6-elongase activity, which are detailed in the application, allowed the identification of sequences from Arabidopsis thaliana and Euglena gracilis, respectively, with corresponding motifs in sequence databases (Genbank, Euglena EST Bank). The sequences were the following:

The Euglena gracilis elongases were cloned as follows:
The Euglena gracilis strain 1224-5/25 was obtained from the Samµlung fur Algenkulturen Gottingen [Gottingen collection of algal cultures] (SAG). For the isolation, the strain was grown for 4 days at 23°C in medium II (Calvayrac R and Douce R, FEBS Letters 7:259-262, 1970) with a photoperiod of 8 h/16 h (light intensity 35 mol s-1 m-2).
Total RNA of a four-day-old Euglena culture was isolated with the aid of the RNAeasy Kit from Qiagen (Valencia, CA, US). poly-A+ RNA (mRNA) was isolated from the total RNA with the aid of oligo-dT-cellulose (Sambrook et al., 1989). The RNA was subjected to reverse transcription with the Reverse Transcription System Kit from Promega, and the cDNA synthesized was cloned into the lambda ZAP vector (lambda ZAP Gold, Stratagene). The cDNA was depackaged in accordance with the manufacturer's instructions to give the plasmid DNA, and clones were partially sequenced for random sequencing. mRNA was isolated from the total RNA with the aid of the PolyATract isolation system (Promega). The mRNA was subjected to reverse

transcription with the Marathon cDNA Amplification Kit (BD Biosciences) and the adaptors were ligated in accordance with the manufacturer's instructions. The cDNA library was then used for the PCR for cloning expression pfasmids by means of 5'- and 3'-RACE (raµl d amplification of cDNA ends).
The Arabidopsis thaliana elongases were cloned as follows:
Starting from the genomic DNA, primers for the two genes were derived at the 51 and the 3' end of the open reading frame.
The method of Chrigwin et a/., (1979) was used for isolating total RNA from A thaliana. Leaves from 21-day-old plants were crushed in liquid nitrogen, treated with disruption buffer and incubated for 15 minutes at 37°C. After centrifugation (10 min, 4°C, 12 000 x g), the RNA in the supernatant was preciµl tated at -20°C for 5 hours using 0.02 volume of 3 M sodium acetate pH 5.0 and 0.75 volume ethanol. After a further centrifugation step, the RNA was taken up in 1 µl of TES per g of starting material, extracted once with one volume of phenol/chloroform and once with one volume of chloroform, and the RNA was preciµl tated with 2.5 M LiCI. Following subsequent centrifugation and washing with 80% ethanol, the RNA was resuspended in water. The cDNA was synthesized in accordance with the method of Sambrook et al. 1989, and an RT-PCR was carried out using the derived primers. The PCR products were cloned into the vector pYES2.1-TOPO (Invitrogen) in accordance with the manufacturer's instructions.
Example 49: Cloning expression plasmids for heterologous expression in yeasts:
To characterize the function of the A. thaliana elongases, the open reading frames of the DNAs in question were cloned downstream of the galactose-inducible GAL1 promoter of pYES2.1/V5-His-TOPO (Invitrogen), giving rise to pAt60 and pAt70.
The Saccharomyces cerevisiae strain 334 was transformed by electroporation (1500 V) with the vector pAt60 and pAt70, respectively. A yeast which was transformed with the blank vector pYES2.1 was used as the control. The transformed yeasts were selected on complete minimal dropout uracil medium (CMdum) agar plates supplemented with 2% glucose. After the selection, in each case three transformants were selected for the further functional expression.
To express the At elongases, precultures of in each case 5 µl of dropout uracil CMdum

liquid medium supplemented with 2% (w/v) raffinose were inoculated with the selected transformants and incubated for 2 days at 30°C, 200 rpm.
5 µl of liquid CMdum medium (without uracil) supplemented with 2% raffinose and 300 ^M of various fatty acids were then inoculated with the precultures to an OD6oo of 0.05. The expression was induced by addition of 2% (w/v) galactose. The cultures were incubated for a further 96 hours at 20°C.
Example 50: Expression of pAt60 and pAt70 in yeasts
Yeasts which had been transformed with the plasmids pYES2.1, pAt60 and pAt70 as described in Example 5 were analyzed as follows:
The yeast cells from the main cultures were harvested by centrifugation (100 x g, 5 min, 20°C) and washed with 100 mM NaHCO3j pH 8.0 to remove residual medium and fatty acids. Starting with the yeast cell sediments, fatty acid methyl esters (FAMEs) were prepared by acid methanolysis. To this end, the cell sediments were incubated for 1 hour at 80°C together with 2 µl of 1 N methanolic sulfuric acid and 2% (v/v) of dimethoxypropane. The FAMEs were extracted twice with petroleum ether (PE). To remove nonderivatized fatty acids, the organic phases were washed in each case once with 2 µl of 100 mM NaHCO3, pH 8.0 and 2 µl of distilled water. Thereafter, the PE phases were dried with Na2SO4, evaporated under argon and taken up in 100 µl of PE. The samples were separated on a DB-23 caµl llary column (30 m, 0.25 mm, 0.25 pm, Agilent) in a Hewlett-Packard 6850 gas chromatograph equipped with flame ionization detector. The conditions for the GLC analysis were as follows: the oven temperature was programmed from 50°C to 250°C with an increment of 5°C/min and finally 10 min at 250°C (holding).
The signals were identified by comparing the retention times with corresponding fatty acid standards (Sigma). The methodology is described for example in Naµl er and Michaelson, 2001, Liµl ds. 36(8):761-766; Sayanova et al., 2001, Journal of Experimental Botany. 52(360): 1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218.
Example 51: Functional characterization of pAt60 and pAt70
The substrate specificity of the elongases At3g06460 and At3g06470 was determined after expression and feeding of various fatty acids (Table 20, Fig. 26). The substrates

which have been fed can be detected in all transgenic yeasts. The transgenic yeasts showed the synthesis of novel fatty acids, the products of the genes At3g06460 and At3g06470, respectively. This means that these genes have been expressed functionally.
Table 20: Elongation of EPA by the elongases At3g06460 and At3g06470,
respectively. Measurement of the yeast extracts after feeding of 250 \M EPA

Figure 26 represents the elongation of 2O;5n-3 by the elongases At3g06470. Example 52: Cloning an elongase from Phaeodactylum tricornutum
Starting from conserved regions in the protein sequences, degenerate primers were constructed with the aid of the elongase genes with ∆6-elongase activity detailed in the application, and these primers were used for searching a Phaeodactylum cDNA library by means of PCR. The following primer sequences were employed:

Nucleotide bases in brackets mean that a mixture of oligonucleotides with in each case one or the other nucleotide base are present.
Construction of the Phaeodactylum cDNA library:
A 2 I culture of P. tricornutum UTEX 646 was grown in f/2 medium (Guillard, R.R.L. 1975. Culture of phytoplankton for feeding marine invertebrates. In Culture of Marine

Invertebrate Animals (Eds. Smith, W.L and Chanley, M.H.), Plenum Press, New York, pp 29-60) for 14 d (= days) at a light intensity of 35 E/cm2. After centrifugation, frozen cells were ground to a fine powder in the presence of liquid nitrogen and resuspended in 2 µl of homogenization buffer (0.33 M sorbitol, 0.3 M NaCI, 10 mM EDTA, 10 mM EGTA, 2% SDS, 2% mercaptoethanol in 0.2 M Tris-CI pH 8.5). After 4 mi of phenol and 2 µl of chloroform had been added, the mixture was shaken vigorously for 15 minutes at 40-50°C. Thereafter, the mixture was centrifuged (10 min x 10 000 g) and the aqueous phase was extracted stepwise with chloroform. Nucleic acids were then preciµl tated by addition of 1/20 volume 4 M sodium hydrogencarbonate solution and centrifuged. The pellet was taken up in 80 mM Tris-borate pH 7.0 and 1 mM EDTA, and the RNA was preciµl tated with 8 M lithium chloride. After centrifugation and washing with 70% strength ethanol, the RNA pellet was taken up in RNase-free water. Poly(A)-RNA was isolated using Dynabeads (Dynal, Oslo, Norway) following the manufacturer's instructions, and the first-strand cDNA synthesis was carried out using µLV-Rtase from Roche (Mannheim). Then, the second-strand synthesis was carried out using DNA polymerase I and Klenow fragment, followed by a digestion with RNaseH. The cDNA was then treated with T4 DNA polymerase, and EcoRI/Xhol adaptors (Pharmacia, Freiburg) were subsequently attached by means of T4 ligase. After digestion with Xhol, phosphorylation and gel separation, fragments greater than 300 bp were ligated into the phage lambda ZAP Express following the manufacturer's instructions (Stratagene, Amsterdam, the Netherlands). Following bulk excision of the cDNA library and plasmid recovery, the plasmid library was transformed into E. coli DH10B cells and employed for the PCR screening.
Using the abovementioned degenerate primers, it was possible to generate the PCR fragment with the sequence number SEQ ID NO: 187.
This fragment was labeled with digoxigenin (Roche, Mannheim) and used as probe for screening the phage library.
With the aid of the sequence SEQ ID NO: 187, it was possible to obtain the gene sequence SEQ ID NO: 183, which constitutes the full-RNA molecule of the Phaeodactylum ∆6-elongase:
Example 53: Cloning expression plasmids for the heterologous expression in yeasts
The relevant primer pairs were selected in such a way that they bore the yeast consensus sequence for highly efficient translation (Kozak, Cell 1986, 44:283-292) next

to the start codon. The amplification of the RELO6 DNAs was carried out in each case using 1 jxl of cDNA, 200 jaM of dNTPs, 2.5 U Advantage polymerase and 100 pmol of each primer in a total volume of 50 \j\. The PCR conditions were as follows: first denaturation for 5 minutes at 95°C, followed by 30 cycles of 30 seconds at 94°C, 1 minute at 55°C and 2 minutes at 72°C, and a last elongation step of 10 minutes at 72°C.
To clone the sequence for the heterologous expression in yeasts, the following oligonucleotides were used for the PCR reaction:

*F=forward primer, R=reverse primer
The PCR products were incubated for 30 minutes at 21 °C with the yeast expression vector pYES2.1-TOPO (Invitrogen) following the manufacturer's instructions. The PCR product (see SEQ ID NO: 192) was ligated into the vector by means of a T overhang and activity of a topoisomerase (Invitrogen). After incubation, E. coli DH5oc cells were transformed. Suitable clones were identified by PCR, the plasmid DNA was isolated by means of Qiagen DNAeasy Kit and verified by sequencing. The correct sequence was then transformed into the Saccharomyces strain INVSd (Invitrogen) by electroporation (1500 V). As a control, the blank vector pYES2.1 was transformed in parallel. The yeasts were subsequently plated onto complete uracil dropout minimal medium supplemented with 2% glucose. Cells which were capable of growing in the medium without uracil thus comprise the corresponding plasmids pYES2.1 and pYES2.1-PtEL06. After the selection, in each case two transformants were selected for further functional expression.
Example 54: Cloning expression plasmids for the seed-specific expression in plants
A further transformation vector based on pSUN-USP is generated for the transformation of plants. To this end, Notl cleavage sites are introduced at the 5' and 3' ends of the coding sequence, using the following primer pair

synthase gene J. Mol. Appl. Genet. 1 (6), 499-511 (1982)). The USP promoter corresponds to nucleotides 1 to 684 (Genbank Accession X56240), where part of the noncoding region of the USP gene is present in the promoter. The promoter fragment which is 684 base pairs in size was amplified by a PCR reaction and standard methods with the aid of a synthesized primer and by means of a commercially available T7 standard primer (Stratagene).
(Primersequence: 5-
GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-31; SEQ ID NO: 151).
The PCR fragment was recut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator. This gave rise to the plasmid with the name pSUN-USP. The construct was used for the transformation of Arabidopsis thaliana, oilseed rape, tobacco and linseed.
Liµl ds were extracted from yeasts and seeds as described for Example 6. Example 55: Expression of PtElo in yeasts
Yeasts which had been transformed with the plasmids pYES2 and pYES2»PtELO6 as in Example 4 were analyzed as follows:
The yeast cells from the main cultures were harvested by centrifugation (100 x g, 5 min, 20°C) and washed with 100 mM NaHCO3) pH 8.0 in order to remove residual medium and fatty acids. Fatty acid methyl esters (FAMEs) were prepared from the yeast cell sediments by acid methanolysis. To this end, the cell sediments were incubated for 1 hour at 80°C with 2 µl of 1N methanolic sulfuric acid and 2% (v/v) dimethoxypropane. The FAMEs were extracted by twice extracting with petroleum ether (PE). To remove nonderivatized fatty acids, the organic phases were washed in each case once with 2 µl of 100 mM NaHCO3, pH 8.0, and 2 µl of distilled water. Thereafter, the PE phases were dried with Na2SO4l evaporated under argon and taken up in 100 pJ of PE. The samples were separated on a DB-23 caµl llary column (30 m, 0.25 mm, 0.25 jam, Agilent) in a Hewlett-Packard 6850 gas chromatograph with flame ionization detector. The conditions for the GLC analysis were as follows: the oven temperature was programmed from 50°C to 250°C with an increment of 5°C/min and finally 10 minutes at 250°C (holding).

The signals were identified by comparing the retention times with corresponding fatty acid standards (Sigma). The methodology is described for example in Naµl er and Michaelson, 2001, Liµl ds. 36(8)761-766; Sayanova et al., 2001, Journal of Experimental Botany. 52(360): 1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218.
Example 56: Functional characterization of PtELO6:
Figure 29 represents the conversion of C18:3∆6'912 and C18.4∆6*9'12'15. The substrates are elongated by in each case two carbon atoms; this results in the fatty acids C20:3A8,ii,i4 and C20:4A8,ii,i4,i7j respectjve|y The substrate specificity of PtELO6 can
be determined after expression and the feeding of different fatty acids (Figure 30). The substrates fed can be detected in large amounts in all of the transgenic yeasts. The transgenic yeasts demonstrated the synthesis of novel fatty acids, the products of the PtElo6 reaction. This means that the gene PtEIO6 has been expressed functionally.
It can be seen from Table 21 that PtElo6 shows a narrow substrate specificity. PtELO6 was only capable of elongating the C18-fatty acids linoleic acid, linolenic acid, y-iinolenic acid and stearidonic acid, but preferred the ω3-desaturated stearidonic acid (see also Figure 30).
Feeding experiment: fatty acids (in bold) were added in each case in amounts of 250 fiM. The underlined fatty acids were formed de novo.

The following fatty acids were fed, but not converted:
• 18:1∆6, 18:1A9, 18:1A11
• 20'2A11'14 20'3A11'14'17 20'3A8'11'14 20'4∆5'8'11'14 20'5∆5'Bl11'14'17
• 22'4∆7'10113116
The yeasts which had been transformed with the vector pYES2-PtELO6 were cultured in minimal medium in the presence of the fatty acids detailed. The fatty acid methyl esters were synthesized by subjecting intact cells to acid methanolysis. Thereafter, the FAMEs were analyzed via GLC. The results shown in Figures 29 and 30 and in Table 19 were thus determined.
Example 57: Cloning expression plasmids for the seed-specific expression in plants
The general conditions described hereinbelow apply to all of the subsequent experiments, unless otherwise specified.
The following are preferably used in accordance with the invention for the examples which follow: Bin 19, pBI101, pBinAR, pGPTV and pCAMBIA. An overview of binary vectors and their use is found in Hellens et al., Trends in Plant Science (2000) 5, 446-451. A pGPTV derivative as described in DE10205607 was used. This vector differs from pGPTV by an additionally inserted Asc\ restriction cleavage site.
Starting point of the cloning procedure was the cloning vector pUC19 (Maniatis et al.). In the first step, the Coniinin promoter fragment was amplified using the following primers:
CnM C 5': gaattcggcgcgccgagctcctcgagcaacggttccggcggtatagagttgggtaattcga CnM C 3': cccgggatcgatgccggcagatctccaccattttttggtggtgat
Composition of the PCR mix (50 µl ):
5.00 µl template cDNA
5.00 M' 10x buffer (Advantage polymerase) + 25mM MgCI2
5.00 µl 2mM dNTP

Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35
The PCR product was first incubated with the restriction enzyme Stu\ for 2 hours at 37°C and then for 12 hours at 25°C with the restriction enzyme Smal. The vector pUC19-Cnl1-C was incubated for 12 hours at 25°C with the restriction enzyme Smal. Thereafter, the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit following the manufacturer's instructions. Thereafter, vector and PCR product were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmid pUC19-Cnl1-C_OCS was verified by sequencing.
In the next step, the Cnl1-B promoter was amplified by PCR using the following primers:
Cnl1-B 5': aggcctcaacggttccggcggtatag
Cnl1-B 31: cccggggttaacgctagcgggcccgatatcggatcccattttttggtggtgattggttct
Composition of the PCR mix (50 µl):
5.00 µl template cDNA
5.00 µl 10x buffer (Advantage polymerase) + 25mM MgCI2
5.00 µl 2 µM dNTP
1.25 µl of each primer (10 pmol/ µl)
0.50 µl Advantage polymerase (Clontech)
PCR reaction conditions:
Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35
The PCR product was first incubated with the restriction enzyme Stu\ for 2 hours at 37°C and then for 12 hours at 25°C with the restriction enzyme Smal. The vector

pUC19-Cnl1-C was incubated for 12 hours at 25°C with the restriction enzyme Smal. Thereafter, the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit following the manufacturer's instructions. Thereafter, vector and PCR product were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmid pUC19-Cnl1C_Cnl1B_OCS was verified by sequencing.
In a further step, the OCS terminator for Cnl1 B was inserted. To this end, the PCR was carried out with the following primers:
OCS2 5': aggcctcctgctttaatgagatatgcgagac OCS2 31: cccgggcggacaatcagtaaattgaacggag
Composition of the PCR mix (50 µl):
5.00 µl template cDNA
5.00 µl 10x buffer (Advantage polymerase) + 25mM MgCI2
5.00 µl 2mM dNTP
1.25 µl of each primer (10 pmol/ µl)
0.50 µl Advantage polymerase (Clontech)
PCR reaction conditions:
Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35
The PCR product was first incubated with the restriction enzyme Stu\ for 2 hours at 37°C and then for 12 hours at 25°C with the restriction enzyme Smal. The vector pUC19-Cnl1C_Cnl1B_OCS was incubated for 12 hours at 25°C with the restriction enzyme Smal. Thereafter, the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit following the manufacturer's instructions. Thereafter, vector and PCR product were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmid pUC19-Cnl1C_Cnl1B_OCS2 was verified by sequencing.

In the next step, the Cnl1-A promoter was amplified by PCR using the following primers:
Cnl1-B 5': aggcctcaacggttccggcggtatagag
Cnl1-B 3': aggccttctagactgcaggcggccgcccgcattttttggtggtgattggt
Composition of the PCR mix (50 µl):
5.00 µl template cDNA
5.00 µl 10x buffer (Advantage polymerase) + 25mM MgCI2
5.00 µl 2mM dNTP
1.25 µl of each primer (10 pmol/( µl)
0.50 µl Advantage polymerase (Clontech)
PCR reaction conditions:
Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35
The PCR product was incubated for 2 hours at 37°C with the restriction enzyme Stul The vector pUC19-Cnl1-C was incubated for 12 hours at 25°C with the restriction enzyme Smal. Thereafter, the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit in accordance with the manufacturer's instructions. Thereafter, vector and PCR product were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmid pUC19-Cnl1C_Cnl1B_Cn!1A_OCS2 was verified by sequencing.
In a further step, the OCS terminator for Cnl1 A was inserted. To this end, the PCR was carried out with the following primers:
OCS2 51: ggcctcctgctttaatgagatatgcga
OCS2 3': aagcttggcgcgccgagctcgtcgacggacaatcagtaaattgaacggaga
Composition of the PCR mix (50 µl): 5.00 µl template cDNA

5.00 µl 10x buffer (Advantage poiymerase) + 25mM MgCI2
5.00 µl 2mM dNTP
1.25 µl of each primer (10 pmol/MO
0.50 µl Advantage poiymerase (Clontech)
PCR reaction conditions:
Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35
The PCR product was first incubated with the restriction enzyme Stu\ for 2 hours at 37°C and then for 2 hours at 37°C with the restriction enzyme HindWl The vector pUC19-CnMC_Cnl1B_Cnl1A_OCS2 was incubated for 2 hours at 37°C with the restriction enzyme Stu\ and for 2 hours at 37°C with the restriction enzyme HindWl Thereafter, the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit following the manufacturer's instructions. Thereafter, vector and PCR product were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmid pUC19-Cnl1C_Cnl1B_CnI1AjDCS3 was verified by sequencing.
In the next step, the plasmid pUC19-Cnl1C_Cnl1B_Cnl1AjDCS3 was used for cloning the ∆6-, ∆5-desaturase and ∆6-elongase. To this end, the ∆6-desaturase from Phytium irregulare (WO02/26946) was amplified using the following PCR primers:
DBDes(µl r) 5': agatctatggtggacctcaagcctggagtg D6Des(µl r) 3': ccatggcccgggttacatcgctgggaactcggtgat
Composition of the PCR mix (50 µl):
5.00 µl template cDNA
5.00 µl 10x buffer (Advantage poiymerase) + 25mM MgCI2
5.00 µl 2mM dNTP
1.25 µl of each primer (10 pmol/ µl)
0.50 µl Advantage poiymerase (Clontech)
PCR reaction conditions:

Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35
The PCR product was first incubated with the restriction enzyme Bgl\\ for 2 hours at 37°C and then for 2 hours at 37°C with the restriction enzyme Nco\. The vector pUC19-Cnl1C_CnMBJDn!1AjDCS3 was incubated for 2 hours at 37°C with the restriction enzyme Bgl\\ and for 2 hours at 37°C with the restriction enzyme Nco\. Thereafter, the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit following the manufacturer's instructions. Thereafter, vector and PCR product were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmid pUC19-Cnl1_d6Des(µl r) was verified by sequencing.
In the next step, the µl asmid µl C19-Cnl1_d6Des(µl r) was used for cloning the ∆5-desaturase from Thraustochytrium ssp. (WO02/26946). To this end, the ∆5-desaturase from Thraustochytrium ssp. was amplified using the following PCR primers:
D5Des(Tc) 5': gggatccatgggcaagggcagcgagggccg D5Des(Tc) 3': ggcgccgacaccaagaagcaggactgagatatc
Composition of the PCR mix (50 µl):
5.00 µl template cDNA
5.00 µl 10x buffer (Advantage polymerase) + 25mM MgCI2
5.00 µl 2mM dNTP
1.25 µl of each primer (10 pmol/µl
0.50 µl Advantage polymerase (Clontech)
PCR reaction conditions:
Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35

The PCR product was first incubated with the restriction enzyme BamHt for 2 hours at 37°C and then for 2 hours at 37°C with the restriction enzyme EcoRS/. The vector pUC19-Cni1_d6Des(µl r) was incubated for 2 hours at 37°C with the restriction enzyme BamH\ and for 2 hours at 37°C with the restriction enzyme EcoRV. Thereafter, the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit following the manufacturer's instructions. Thereafter, vector and PCR product were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmid pUC19-Cnl1_d6Des(µl r)_d5Des(Tc) was verified by sequencing.
In the next step, the plasmid pUC19-CnI1_d6Des(µl r)_d5Des(Tc) was used for cloning the ∆6-elongase from Physcomitrella patens (WO01/59128), to which end an amplification with the following PCR primers was carried out:
D6Elo(Pp) 5': gcggccgcatggaggtcgtggagagattctacggtg D6Elo(Pp) 3': gcaaaagggagctaaaactgagtgatctaga
Composition of the PCR mix (50 µl):
5.00 µl template cDNA
5.00 µl 10x buffer (Advantage poiymerase) + 25mM MgCI2
5.00 µl 2mM dNTP
1.25 yl of each primer (10 pmol/µl )
0.50 µl Advantage poiymerase (Clontech)
PCR reaction conditions:
Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35
The PCR product was first incubated with the restriction enzyme Not\ for 2 hours at 37°C and then for 2 hours at 37°C with the restriction enzyme Xbal The vector pUC19-i Cnl1_d6Des(µl r)_d5Des(Tc) was incubated for 2 hours at 37°C with the restriction enzyme Notl and for 2 hours at 37°C with the restriction enzyme Xbal. Thereafter, the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the

Qiagen Gel Purification Kit following the manufacturer's instructions. Thereafter, vector and PCR product were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmid pUC19-Cnl1_d6Des(µl r)_d5Des(Tc)_D6Elo(Pp) was verified by sequencing.
The binary vector for the plant transformation was generated starting from pUC19-Cnl1_d6Des(µl r)_d5Des(Tc)JD6Elo(Pp). To this end, pUC19-Cnl1_d6Des(µl r)_d5Des(Tc)JD6Elo(Pp) was incubated for 2 hours at 37°C with the restriction enzyme Asc\. The vector pGPTV was treated in the same manner. Thereafter, the fragment from pUC19-Cnl1_d6Des(µl r)_d5Des(TcLD6Elo(Pp) and the cut pGPTV vector were separated by agarose gel electrophoresis and the relevant DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit in accordance with the manufacturer's instructions. Thereafter, vector and PCR product were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmid pGPTV-CnI1_d6Des(µl r)jd5Des(Tc)_D6Elo(Pp) was verified by sequencing.
A further construct, pGPTV-Cnl1_d6Des(µl rLd5Des(TcLD6Elo(PpLD12Des(Co), was used. To this end, an amplification was performed starting from pUC19-Cnl1C_OCS, using the following primers:
Cnl1 J3CS 5': gtcgatcaacggttccggcggtatagagttg Cnl1_OCS 3': gtcgatcggacaatcagtaaattgaacggaga
Composition of the PCR mix (50 µl):
5.00 µl template cDNA
5.00 µl 10x buffer (Advantage polymerase) + 25mM MgCI2
5.00 µl 2mM dNTP
1.25 µl of each primer (10 pmol/µl
0.50 µl Advantage polymerase (Clontech)
PCR reaction conditions:
Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35

The PCR product was incubated for 2 hours at 37°C with the restriction enzyme Sa/1. The vector pUC19 was incubated for 2 hours at 37°C with the restriction enzyme Sa/I. Thereafter, the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit in accordance with the manufacturer's instructions. Thereafter, vector and PCR product were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmid pUC19-Cnl1_OCS was verified by sequencing.
In a further step, the ∆12-desaturase gene from Calendula officinalis (WO01/85968) was cloned into pUC19-Cnl1_OCS. To this end, d12Des(Co) was amplified using the following primers:
D12Des(Co) 5': agatctatgggtgcaggcggtcgaatgc D12Des(Co) 31: ccatggttaaatcttattacgatacc
Composition of the PCR mix (50 µl ):
5.00 µl template cDNA
5.00 µl 10x buffer (Advantage polymerase) + 25mM MgCI2
5.00 µl 2mM dNTP
1.25 µl of each primer (10 pmol/µl )
0.50 µl Advantage polymerase (Clontech)
PCR reaction conditions:
Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35
The PCR product was incubated for 2 hours at 37°C with the restriction enzyme BglW and subsequently for 2 hours at the same temperature with A/col. The vector pUC19-Cnl1_OCS was incubated in the same manner. Thereafter, the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit in accordance with the manufacturer's instructions. Thereafter, vector and PCR product were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmid pUC19-Cn!1_D12Des(Co) was verified by sequencing.

The plasmid pUC19-Cnl1_D12Des(Co) and the plasmid pUC19-Cnl1_d6Des(µl r)_d5Des(TcLD6Elo(Pp) were incubated for 2 hours at 37°C with the restriction enzyme Sa/I. Thereafter, the vector fragment and the vector were separated by agarose gel electrophoresis and the relevant DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit in accordance with the manufacturer's instructions. Thereafter, vector and vector fragment were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmid pUC19-Cnl1_d6Des(µl r)_d5Des(Tc)_D6Elo(Pp)_D12Des(Co) was verified by sequencing.
The binary vector for the plant transformation was generated starting from pUC19-CnM_d6Des(µl rLd5Des(Tc)_D6Elo(PpLD12Des(Co). To this end, pUC19-Cnl1_d6Des(µl r)_d5Des(Tc)_D6Elo(Pp)_D12Des(Co) was incubated for 2 hours at 37°C with the restriction enzyme Ascl. The vector pGPTV was treated in the same manner. Thereafter, the fragment from pUC19-
CnM_d6Des(µl r)_d5Des(TcLD6Elo(Pp)_D12Des(Co) and the cut pGPTV vector were separated by agarose gel electrophoresis and the relevant DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit in accordance with the manufacturer's instructions. Thereafter, vector and PCR product were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmid pGPTV-Cnh_d6Des(µl r)_d5Des(Tc)_D6Elo(Pp)_D12Des(Co) was verified by sequencing.
A further vector which is suitable for the transformation of plants is pSUN2. To increase the number of expression cassettes present in the vector to more than four, this vector was used in combination with the Gateway System (Invitrogen, Karlsruhe). To this end, the Gateway cassette A was inserted into the vector pSUN2 in accordance with the manufacturer's instructions as described hereinbelow:
The pSUN2 vector (1 jig) was incubated for 1 hour with the restriction enzyme EcoRV at 37°C. Thereafter, the Gateway cassette A (Invitrogen, Karlsruhe) was ligated into the cut vector by means of the Raµl d Ligation Kit from Roche, Mannheim. The resulting plasmid was transformed into E. coii DB3.1 cells (Invitrogen). The isolated plasmid pSUN-GW was subsequently verified by sequencing.
In the second step, the expression cassette was excised from pUC19-Cnl1_d6Des(µl r)_d5Des(Tc)_D6Elo(Pp)_D12Des(Co) by means of Ascl and ligated

into the vector pSUN-GW, which had been treated in the same manner. The resulting plasmid pSUN-4G was used for further gene constructs.
To this end, a pENTR clone was first modified in accordance with the manufacturer's instructions (Invitrogen). The plasmid pENTRIA (Invitrogen) was incubated for 1 hour at 37°C with the restriction enzyme Ecorl, subsequently treated for 30 minutes with Klenow enzyme and with one 1 jaM dNTP mix, and the AscI adaptor (5'-ggcgcgcc; phosphorylated at the 5' terminus, double-stranded) was then ligated into the vector pENTRIA. Into this modified, genes were stepwise inserted into the Cnl cassette as described above and transferred into the pENTR vector via AscI.
The gene TL16y2 from Thraustochytrium ssp. (SEQ ID NO: 83) was transferred into the pSUN-4G vector in the abovedescribed manner:
In the next step, the plasmid pUC19-Cnl1C_CnMB_Cnl1A_OCS3 was used for cloning the ∆5-elongase TL16y2. To this end, the ∆5-elongase from Thraustochytrium ssp. was amplified using the following PCR primers:
TL16y2 5': agatct atggacgtcgtcgagcagca TL16y2 3': ccatggcccggg agaagcagaagaccatctaa
Composition of the PCR mix (50 µl):
5.00 µl template cDNA
5.00 µl 10x buffer (Advantage polymerase) + 25mM MgCI2
5.00 µl 2mM dNTP
1.25 µl of each primer (10 pmol/µl
0.50 µl Advantage polymerase (Clontech)
PCR reaction conditions:
Annealing temperature: 1 min 55°C Denaturation temperature: 1 min 94°C Elongation temperature: 2 min 72°C Number of cycles: 35
The PCR product was first incubated for 2 hours at 37°C with the restriction enzyme Bgftl and then for 2 hours at 37°C with the restriction enzyme A/col. The vector pUC19-Cnl1C_Cnl1B_CnMA_OCS3 was incubated for 2 hours at 37°C with the restriction

enzyme Bgl\\ and for 2 hours at 37°C with the restriction enzyme A/col. Thereafter, the PCR product and the cut vector were separated by agarose gel electrophoresis and the relevant DNA fragments were excised. The DNA was purified by means of the Qiagen Gel Purification Kit in accordance with the manufacturer's instructions. Thereafter, vector and PCR product were ligated. The Raµl d Ligation Kit from Roche was used for this purpose. The resulting plasmid pUC19-Cnl1_TL16y2 was verified by sequencing. Thereafter, the cassette was excised using AscI and ligated into an Ascl-pretreated pENTR vector. The resulting plasmid pENTR-Cnl1_TL16y2 was then incubated with the vector pSUN-4G in a recombination reaction in accordance with the manufacturer's instructions (Invitrogen). The product gave the vector pSUN-5G, which was used for the transformation of plants.
In a further step, the construct pSUN-8G was generated using the above-described methodology. To this end, 5' and 3' primers for the genes SEQ ID 41, 53, 87 and 113 with the above-described restriction cleavage sites and the first and in each case last 20 nucleotides of the open reading frame were generated, amplified under the standard conditions (see above) and ligated into the vector pENTR-Cnl.
A recombination reaction with the vector pSUN-4G gave rise to the construct pSUN-8G. This vector too was employed for the transformation of plants.
Example 58: Generation of transgenic plants
a) Generation of transgenic Indian mustard plants. The protocol for the
transformation of oilseed rape plants was used (modification of the method of Moloney et al., 1992, Plant Cell Reports, 8:238-242)
To generate transgenic plants, the binary vectors pGPTV-
Cnl1Md6Des(µl r)_d5Des(TcLD6Elo(Pp)_D12Des(Co), pSUN-5G and pSUN-8G which had been generated were transformed into Agrobacterium tumefaciens C58C1:pGV2260 (Deblaere et al., 1984, Nucl. Acids Res. 13, 4777-4788). To transform Indian mustard plants, a 1:50 dilution of an overnight culture of a positively transformed agrobacterial colony in Murashige-Skoog medium (Murashige and Skoog 1962 Physiol. Plant. 15, 473) supplemented with 3% sucrose (3MS medium) was used. Petioles or hypocotyls of freshly germinated sterile plants (in each case approx. 1 cm2) were incubated for 5-10 minutes with a 1:50 agrobacterial dilution in a Petri dish. This is followed by 3 days of coincubation in the dark at 25°C on 3MS medium supplemented with 0.8% Bacto agar. Cultivation was subsequently continued at 16

hours light/8 hours dark and in a weekly rhythm on MS medium supplemented with 500 mg/l of Claforan (cefotaxime-sodium), 50 mg/l kanamycin, 20 pM benzylaminopurine (BAP) and 1.6 g/l glucose. Growing shoots were transferred to MS medium supplemented with 2% sucrose, 250 mg/l Claforan and 0.8% Bacto agar. If no roots had formed after three weeks, 2-indolebutyric acid was added to the medium for rooting, to act as growth hormone.
Regenerated shoots were maintained on 2MS medium supplemented with kanamycin and Claforan, after rooting, transferred into soil and, after cultivation, grown for two weeks in a controlled-environment cabinet or in a greenhouse, allowed to flower, mature seeds were harvested and studied for elongase expression such as ∆6-elongase activity or ∆5- or ∆6-desaturase activity by means of liµl d analyses. In this manner, lines with elevated contents of C20- and C22-polyunsaturated fatty acids were identified.
Transgenic oilseed rape plants were also generated successfully using this protocol, b) Generation of transgenic linseed plants
The transgenic linseed plants can be generated for example by the method of Bell et al., 1999, In Vitro Cell. Dev. Biol.-Plant. 35(6): 456-465 by means of particle bombardment. Agrobacteria-mediated transformations can be carried out for example by the method of µlynarova et al. (1994), Plant Cell Report 13: 282-285.
Example 59: Liµl d extraction from seeds:
The effect of the genetic modification in plants on the production of a desired compound (such as a fatty acid) can be determined by growing the modified plant under suitable conditions (such as those described above) and analyzing the medium and/or the cellular components for the elevated production of the desired product (i.e. of the liµl ds or a fatty acid). These analytical techniques are known to the skilled worker and comprise spectroscopy, thin-layer chromatography, various types of staining methods, enzymatic and microbiological methods and analytical chromatography such as high-performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, pp 89-90 and pp 443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987) "Applications of HPLC in Biochemistry" in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol. 3, Chapter III: "Product recovery and purification", pp 469-714,

VCH: Weinheim; Belter, PA, et al. (1988) Bioseparations: downstream processing for Biotechnology, John Wiley and Sons; Kennedy, J.F., and Cabral, J.M.S. (1992) Recovery processes for biological Materials, John Wiley and Sons; Shaeiwitz, JA, and Henry, J.D. (1988) Biochemical Separations, in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3; Chapter 11, pp 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publications).
In addition to the abovementioned methods, plant liµl ds are extracted from plant material as described by Cahoon et al. (1999) Proc. Natl. Acad. ScL USA 96 (22):12935-12940 and Browse et al. (1986) Analytic Biochemistry 152:141-145. The qualitative and quantitative analysis of liµl ds or fatty acids is described by Christie, William W., Advances in Liµl d Methodology, Ayr/Scotland: Oily Press (Oily Press Liµl d Library; 2); Christie, William W., Gas Chromatography and Liµl ds. A Practical Guide -Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307 pp (Oily Press Liµl d Library; 1); "Progress in Liµl d Research, Oxford: Pergamon Press, 1 (1952) -16 (1977) under the title: Progress in the Chemistry of Fats and Other Liµl ds CODEN.
In addition to measuring the end product of the fermentation, it is also possible to analyze other components of the metabolic pathways which are used for the production of the desired compound, such as intermediates and by-products, in order to determine the overall production efficiency of the compound. The analytical methods comprise measuring the amount of nutrients in the medium (for example sugars, hydrocarbons, nitrogen sources, phosphate and other ions), measuring the biomass composition and the growth, analyzing the production of conventional metabolites of biosynthetic pathways and measuring gases which are generated during the fermentation. Standard methods for these measurements are described in Applied Microbial Physiology; A Practical Approach, P.M. Rhodes and P.F. Stanbury, Ed., IRL Press, pp 103-129; 131-163 and 165-192 (ISBN: 0199635773) and references cited therein.
One example is the analysis of fatty acids (abbreviations: FAME, fatty acid methyl ester; GC-MS, gas liquid chromatography/mass spectrometry; TAG, triacylglycerol; TLC, thin-layer chromatography).
Unambiguous proof of the presence of fatty acid products can be obtained by analyzing recombinant organisms using standard analytical methods: GC, GC-MS or TLC, as described on several occasions by Christie and the references therein (1997, in: Advances on Liµl d Methodology, Fourth Edition: Christie, Oily Press, Dundee, 119-169;

1998, Gaschromatographie-Massenspektrometrie-Verfahren [Gas chromatography/mass spectrometry methods], Liµl de 33:343-353).
The material to be analyzed can be disrupted by sonication, grinding in a glass mill, liquid nitrogen and grinding or via other applicable methods. After disruption, the material must be centrifuged. The sediment is resuspended in distilled water, heated for 10 minutes at 100°C, cooled on ice and recentrifuged, followed by extraction for 1 hour at 90°C in 0.5 M sulfuric acid in methanol with 2% dimethoxypropane, which leads to hydrolyzed oil and liµl d compounds, which give transmethylated liµl ds. These fatty acid methyl esters are extracted in petroleum ether and finally subjected to a GC analysis using a caµl llary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 jj.m, 0.32 mm) at a temperature gradient of between 170°C and 240°C for 20 minutes and 5 minutes at 240°C. The identity of the resulting fatty acid methyl esters must be defined using standards which are available from commercial sources (i.e. Sigma).
Plant material is initially homogenized mechanically by crushing in a pestle and mortar to make it more amenable to extraction.
This is followed by heating at 100°C for 10 minutes and, after cooling on ice, by resedimentation. The cell sediment is hydrolyzed for 1 hour at 90°C with 1 M methanolic sulfuric add and 2% dimethoxypropane, and the liµl ds are transmethylated. The resulting fatty acid methyl esters (FAMEs) are extracted in petroleum ether. The extracted FAMEs are analyzed by gas liquid chromatography using a caµl llary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) and a temperature gradient of from 170°C to 240°C in 20 minutes and 5 minutes at 240°C. The identity of the fatty acid methyl esters is confirmed by comparison with corresponding FAME standards (Sigma). The identity and position of the double bond can be analyzed further by suitable chemical derivatization of the FAME mixtures, for example to give 4,4-dimethoxyoxazolin derivatives (Christie, 1998) by means of GC-MS.
Example 60: Analysis of the seeds from the transgenic plants which have been
generated
Analogously to Example 59, the seeds of the plants which had been transformed with the constructs pGPTV-Cnl1_d6Des(µl rLd5Des(TcLD6Elo(Pp)_D12Des(Co), pSUN-5G and pSUN-8G were analyzed. Figure XX shows the fatty acid spectrum of seeds with the construct pGPTV-Cnl1_d6Des(µl r)_d5Des(TcLD6Elo(Pp)_D12Des(Co). In

comparison with control plants which were not transformed (wild-type control, WT), a pronounced change in the fatty acid spectrum was observed. It was thus possible to demonstrate that the transformed genes are functional. Table 22 comµl les the results of Figure 32.
Table 22:

Here, the analysis of the seeds with the construct pSUN-5G reveals lines with a pronounced increase in the arachidonic acid content in comparison with the construct pGPTV-Cnl1_d6Des(µl rLd5Des(TcLD6Elo(Pp)_D12Des(Co). In this context, lines with up to 25% ARA were obtained. The additional elongase (TL16y2) must be responsible for this effect (Figure 31, pSUN-5G). The results from this line are comµl led in Table 23.

Example 61: Detection of DHA in seeds of transgenic Indian mustard plants.
Seeds of plants which had been generated with the construct pSUN-8G as described in Example 58 were analyzed as described in Example 59. Besides the LCPUFAs arachidonic acid and eicosapentaenoic acid, docosahexaenoic acid, the product after conversion by the ∆4-desaturase from Thraustochytrium and ∆5-elongases from Onchorynchis mykiss and Ostreococcus tauri, was also detected in these seeds. Figure 32 shows the chromatogram with the modified fatty acid spectrum in comparison with an untransformed control plant. The results of several measurements are comµl led in Table 24.

Table 24 shows the fatty acid analysis of transgenic seeds which have been transformed with the construct pSUN-8G.
In this experiment, the synthesis of docosahexaenoic acid in seeds was demonstrated for the first time. While the synthesis of DHA in higher plants has been described, for example in WO 2004/071467, the synthesis has not been demonstrated for seeds, only for an embryogenic cell culture.
Equivalents:
Many equivalents of the specific embodiments according to the invention described herein can be seen or found by the skilled worker by simple routine experiments. These equivalents are intended to be included in the patent claims.

We claim:
1. A process for the production of compounds of the general formula I

in the seed of transgenic plants with a content of at least 20% by weight based on the total liµl d content, which comprises the following process steps;
a) introducing, into the organism, at least one nucleic acid sequence which encodes a ∆9-elongase and ∆6-desaturase activity, and
b) introducing, into the organism, at least one nucleic acid sequence which encodes a ∆8-desaturase and ∆6-elongase activity, and
c) introducing, into the organism, at least one nucleic acid sequence which encodes a ∆5-desaturase activity, and
d) introducing, into the organism, at least one nucleic acid sequence which encodes a ∆5-elongase activity, and
e) introducing, into the organism, at least one nucleic acid sequence which encodes a ∆4-desaturase activity, and
where the variables and substituents in formula I have the following meanings:
R1 = hydroxyl, coenzyme A (thioester), lysophosphatidylcholine,
lysophosphatidylethanolamine, lysophosphatidylglycerol, lyso-diphosphatidylglycerol, lysophosphatidylserine, lysophosphatidylinositol, sphingo base or a radical of the general formula II

33 Fig/202 seq

Rz = hydrogen, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysodiphosphatidylglycerol, lysophosphatidylserine, lysophosphatidylinositol or saturated or unsaturated C2"C24-alkylcarbonyl,
R3 = hydrogen, saturated or unsaturated C2-C24-alkylcarbonyl, or R2 and R3 independently of one another are a radical of the general formula la:

in which
n = 2, 3, 4, 5, 6, 7 or 9, m = 2, 3, 4, 5 or 6 and p = 0 or 3.
2. The process according to claim 1, wherein the variables n, m and p have the following meanings:
n = 2, 3 or 5, m = 4, 5 or 6 and p = 0 or 3.
3. The process according to claim 1 or 2, wherein, in formula l,m = 4, n = 3, p = 3 and the compound is arachidonic acid and/or m = 5,n = 3,p = 0 and the compound is eicosapentaenoic acid and/or m = 5,n = 5, p = 0 and the compound is docosapentaenoic acid and/or m = 6, n = 3, p = 0 and the compound is docosahexaenoic acid.
4. The process according to claims 2 to 3, wherein, in the seed of the transgenic plant, the content of all compounds of the formula I together amounts to at least 27% by weight based on the total liµl d content.
5. The process according to claims 2 to 3, wherein, in the seed of the transgenic plant, the docosahexaenoic acid content amounts to at least 1% by weight based on the total liµl d content.
6. The process according to claims 1 to 5, wherein the nucleic acid sequences

consisting of:
a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 87 or SEQ ID NO: 105, or
b) nucleic acid sequences which, as the result of the degeneracy of the genetic code, can be derived from the amino acid sequence shown in SEQ ID NO: 88 or SEQ ID NO: 106, or
c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 87 or SEQ ID NO: 105, which encode polypeptides with at least 60% identity at the amino acid level with SEQ ID NO: 88 or SEQ ID NO: 106 and which have ω3-desaturase activity
is additionally introduced into the transgenic plant.
8. The process according to claims 1 to 7, wherein a nucleic acid sequence which encodes polypeptides with ∆12-desaturase activity, selected from the group consisting of:
a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 107, SEQ ID NO: 109 or SEQ ID NO: 195, or
b) nucleic acid sequences which, as the result of the degeneracy of the genetic code, can be derived from the amino acid sequence shown in SEQ ID NO: 108, SEQ ID NO: 110 or SEQ ID NO: 196, or
c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 107, SEQ ID NO: 109 or SEQ ID NO: 195, which encode polypeptides with at least 60% identity at the amino add level with SEQ ID NO: 108, SEQ ID NO: 110 or SEQ ID NO: 196 and which have ∆12-desaturase activity
is additionally introduced into the transgenic plant.
9. The process according to claims 1 to 8, wherein a nucleic acid sequence which encodes proteins of the biosynthetic pathway of the fatty acid or liµl d metabolism selected from the group acyl-CoA dehydrogenase(s), acyl-ACP [= acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid

acyltransferase(s), acyl-CoA:lysophospholiµl d acyltransferase(s), fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglyceroi lipases, allenoxide syntheses, hydroperoxide lyases or fatty acid elongase(s) is additionally introduced into the transgenic plant.
10. The process according to claims 1 to 9, wherein the substituents R2 or R3 independently of one another are saturated or unsaturated C18-C22- alkylcarbonyl.
11. The process according to claims 1 to 10, wherein the substituents R2 or R3 independently of one another are unsaturated C18-, C20- or C22-alkylcarbonyl with at least two double bonds.
12. The process according to claims 1 to 11, wherein the transgenic plant is selected from the group of an oil-producing plant, a vegetable plant or an ornamental.
13. The process according to claims 1 to 12, wherein the transgenic organism is a transgenic plant selected from the group of the plant families: Anacardiaceae, Asteraceae, Boraginaceae, Brassicaceae, Cannabaceae, Compositae, Cruciferae, Cucurbitaceae, Elaeagnaceae, Euphorbiaceae, Fabaceae, Geraniaceae, Gramineae, Leguminosae, Linaceae, Malvaceae, Moringaceae, Marchantiaceae, Onagraceae, Olacaceae, Oleaceae, Papaveraceae, µl peraceae, Pedaliaceae, Poaceae or Solanaceae.
14. The process according to claims 1 to 13, wherein the compounds of the general formula I are isolated from the organism in the form of their oils, liµl ds or free fatty acids.
15. A process for the production, in transgenic plants, of compounds of the general formula I according to claim 1, comprising:
a) introducing, into a plant, at least one nucleic acid sequence which encodes a polypeptide with a ∆6-desaturase activity and is selected from the group consisting of:

i) a nucleic acid sequence with the sequence shown in SEQ ID
NO: 193 or SEQ ID NO: 201, ii) nucleic acid sequences which encode the amino acid sequence
shown in SEQ ID NO: 194 or SEQ ID NO: 202, iii) nucleic acid sequences which hybridize under stringent
conditions with the complementary strand of the nucleic acid
sequence shown in SEQ ID NO: 193 or SEQ ID NO: 201, and iv) nucleic acid sequences which have at least 60% identity with the
sequence shown in SEQ ID NO: 193 or SEQ ID NO: 201,
b) introducing, into a plant, at least one nucleic acid sequence which encodes a polypeptide with a ∆6-elongase activity and is selected from the group consisting of:
i) a nucleic acid sequence with the sequence shown in SEQ ID
NO: 27 or SEQ ID NO: 199, ii) nucleic acid sequences which encode the amino acid sequence
shown in SEQ ID NO: 28 or SEQ ID NO: 200, iii) nucleic acid sequences which hybridize under stringent
conditions with the complementary strand of the nucleic acid
sequence shown in SEQ ID NO: 27 or SEQ ID NO: 199, and iv) nucleic acid sequences which have at least 60% identity with the
sequence shown in SEQ ID NO: 27 or SEQ ID NO: 199, and
c) introducing, into a plant, at least one nucleic acid sequence which encodes a polypeptide with a ∆5-desaturase activity and is selected from the group consisting of:
i) a nucleic acid sequence with the sequence shown in SEQ ID
NO: 11, ii) nucleic acid sequences which encode the amino acid sequence
shown in SEQ ID NO: 12, iii) nucleic acid sequences which hybridize under stringent
conditions with the complementary strand of the nucleic acid
sequence shown in SEQ ID NO: 11, and iv) nucleic acid sequences which have at least 60% identity with the
sequence shown in SEQ ID NO: 11, where the variables and substituents in formula I have the meaning given in

claim 1.
16. The process according to claim 15, where the substituents R2 or R3 independently of one another are saturated or unsaturated C10-C22-alkylcarbonyl.
17. The process according to claim 15 or 16, wherein the substituents R2 or R3 independently of one another are unsaturated C18-, C20- or C22-alkylcarbonyl with at least two double bonds.
18. The process according to claims 15 to 17, wherein a nucleic acid sequence which encodes a polypeptide with a +∆12-desaturase activity is additionally introduced into the plant.
19. The process according to claim 18, wherein the nucleic acid sequence is selected from the group consisting of:

20. The process according to claim 18, wherein the ∆12-desaturase is expressed under the control of a seed-specific promoter.
21. The process according to claims 15 to 20, wherein a nucleic acid sequence which encodes a polypeptide with a ∆5-elongase activity is additionally introduced into the plant.
22. The process according to claim 21, wherein the nucleic acid sequence is selected from the group consisting of:

23. The process according to claim 21, wherein the ∆5-elongase is expressed under the control of a seed-specific promoter.
24. The process according to claims 12 to 24, wherein all nucleic acid sequences

are introduced into the plants on a shared recombinant nucleic acid molecule.
25. The process according to claim 24, wherein each nucleic acid sequence is under the control of its own promoter.
26. The process according to claim 25, wherein the own promoter is a seed-specific promoter.
27. The process according to claims 15 to 26, wherein, in formula I, m = 4, n = 3, p = 3 and the compound is arachidonic acid and/or m = 5, n = 3, p = 0 and the compound is eicosapentaenoic acid and/or m = 6,n = 3, p = 0 and the compound is docosahexanoic acid.
28. The process according to claims 15 to 27, wherein the plant is an oil seed plant or oil fruit plant.
29. The process according to claim 28, wherein the plant is selected from the group consisting of soybean, peanut, oilseed rape, canola, linseed, evening primrose, mullein, thistle, hazelnut, almond, macadamia, avocado, bay, wild roses, pumpkin/squash, pistachios, sesame, sunflower, safflower, borage, maize, poppy, mustard, hemp, castor-oil plant, olive, Calendula, Punica, oil palm, walnut and coconut.
30. The process according to claim 28 or 29, wherein the plant is Brassicajuncea.
31. The process according to claims 15 to 30, wherein the compounds of the formula I are obtained from the plant in the form of their oils, lipids and free fatty acids.
32. The process according to claim 31, wherein unsaturated or saturated fatty acids are liberated from the compounds of the formula I.
33. The process according to claim 32, wherein the liberation is effected by alkaline hydrolysis or enzymatic cleavage.
34. The process according to claims 15 to 33, wherein the arachidonic add concentration amounts to at least 25% based on the total lipid content of the transgenic plant.

35. The process according to claims 15 to 33, wherein the eicosapentaenoic acid concentration amounts to at least 15% based on the total lipid content of the transgenic plant.
36. An oil, lipid or fatty acid or a fraction thereof, obtained by a process according to one of the preceding claims.
37. The use of a ∆12-elongase, a ∆6-desaturase, a ∆5-desaturase, a ∆6-elongase and ∆5-elongase as defined in claim 15, 18 or 21, for the production of compounds of the formula I according to claim 1.
38. A recombinant nucleic acid molecule comprising:

a) one or more copies of a promoter which is active in plant cells, preferably in seed cells,
b) at least one nucleic add sequence as defined in claim 15 which encodes a ∆6-desaturase activity,
c) at least one nucleic acid sequence as defined in claim 15 which encodes a ∆5-desaturase activity,
d) at least one nucleic acid sequence as defined in claim 15 which encodes a ∆6-elongase activity, and
e) one or more copies of a terminator sequence.

coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, ailenoxide synthases, hydroperoxide lyases and fatty acid elongase(s).
42. The recombinant nucleic acid molecule according to any of claims 38 to 41, additionally comprising biosynthesis genes of the fatty acid or lipid metabolism, selected from the group consisting of ∆4-desaturase, ∆8-desaturase, ∆9- desaturase or ∆9-elongase.
43. A transgenic plant comprising a recombinant nucleic acid molecule according to any of claims 38 to 42 or comprising the nucleic acid sequences defined in claim 15 and, if appropriate, additionally those defined in claim 18 or 21.
44. The process according to claims 15 to 35, wherein the compounds of the general formula I are isolated from the organism in the form of their oils, lipids or free fatty acids.
45. An oil, lipid or fatty acid or a fraction thereof, produced by the process according to any of claims 15 to 35.
46. An oil, lipid or fatty acid composition which comprises PUFAs produced by a process according to any of claims 1 to 14 and which is derived from transgenic plants.
47. A process for the production of oils, lipids or fatty acid compositions by mixing oils, lipids or fatty acids according to claim 45 or oil, iipid or fatty acid compositions according to claim 46 with animal or microbial oils, lipids or fatty acids.
48. The use of oils, lipids or fatty acids according to claim 45 or oil, lipid or fatty acid compositions according to claim 46 or oils, lipids or fatty acid compositions produced according to claim 46 in feed, foodstuffs, cosmetics or Pharmaceuticals.
49. An isolated nucleic acid sequence which encodes polypeptides with ∆5- elongase activity and which has the sequence shown in SEQ ID NO: 197.
50. An isolated nucleic acid sequence which encodes polypeptides with ∆6-

elongase activity and which has the sequence shown in SEQ ID NO: 199.
51. An isolated nucleic acid sequence which encodes polypeptides with ∆6- desaturase activity and which has the sequence shown in SEQ ID NO: 201.
52. A gene construct comprising an isolated nucleic acid according to any of claims 49 to 51, wherein the nucleic acid is linked functionally to one or more regulatory signals.
53. The gene construct according to claim 53, wherein the nucleic acid construct comprises additional biosynthesis genes of the fatty acid or lipid metabolism selected from the group acyl-CoA dehydrogenase(s), acyl-ACP [= acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyltransferase(s), acyl-CoA:lysophospholipid acyltransferase(s), fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allenoxide synthases, hydroperoxide lyases or fatty acid elongase(s).
54. A vector comprising a nucleic acid according to claim 50 or 51 or a gene construct according to claim 52 or 53.
55. A transgenic plant comprising at least one nucleic acid according to claim 50 or 51 or a gene construct according to claim 52 or 53 or a vector according to claim 54.
Dated this 27 day of September 2006